Compositions and methods for identifying agents which modulate PTEN function and PI-3 kinase pathways

ABSTRACT

Methods are provided for the identification, biochemical characterization and therapeutic use of agents which impact PTEN, p53, PI-kinase and AKT mediated cellular signaling.

[0001] This invention claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Nos. 60/208,437 and 60/274,167 filed May 30,2000 and Mar. 8, 2001 respectively. The entire disclosures of each ofthe above-identified applications is incorporated by reference herein.

[0002] Pursuant to 35 U.S.C. §202(c), it is hereby acknowledged that theU.S. Government has certain rights in the invention described herein,which was made in part with funds from the National Institutes ofHealth, Grant Nos: RO1CA75637.

FIELD OF THE INVENTION

[0003] This invention relates to the treatment of neoplastic disease andother pathological conditions characterized by cellularhyperproliferation and loss of regulated growth and motility. Morespecifically, this invention provides methods for the identification andcharacterization of agents which modulate PTEN, PI-3 kinase and AKTactivity.

BACKGROUND OF THE INVENTION

[0004] Various scientific and scholarly articles are cited throughoutthe specification. These articles are incorporated by reference hereinto describe the state of the art to which this invention pertains.

[0005] The reversible phosphorylation of proteins and lipids is criticalto the control of signal transduction in mammalian cells and isregulated by kinases and phosphatases (Hunter 1995). The product of thetumor suppressor gene PTEN/MMAC (hereafter termed PTEN) was identifiedas a dual specificity phosphatase and has been shown to dephosphorylateinositol phospholipids in vivo (Li et al Science 1997, Steck et al 1997,Li et al Cancer Res 1997,Myers et al, 1997, Myers et al 1998, Maehama etal, 1998, Stambolic et al 1998, Wu et al 1998). The PTEN gene, which islocated on the short arm of chromosome 10 (10q23), is mutated in 40-50%of high grade gliomas as well as many other tumor types, including thoseof the prostate, endometrium, breast, and lung (Li et al, Science 1997,Steck et al 1997, Maier et al 1998). In addition, PTEN is mutated inseveral rare autosomal dominant cancer predisposition syndromes,including Cowden disease, Lhermitte-Duclos disease and Bannayan-Zonanasyndrome (Liaw et al 1997, Myers et al AJHG 1997, Maehama et al TCB1999, Cantley and Neel 1999). Furthermore, the phenotype ofPTEN-knockout mice revealed a requirement for this phosphatase in normaldevelopment and confirmed its role as a tumor suppressor (Podsypanina etal PNAS 1999, Suzuki et al Curr Biol 1998, Di Christofano et al Nat Gen1998).

[0006] PTEN is a 55 kDa protein comprising an N-terminal catalyticdomain, identified as a segment with homology to the cytoskeletalprotein tensin and containing the sequence HC(X)₅R, which is thesignature motif of members of the protein tyrosine phosphatase family,and a C-terminal C2 domain with lipid-binding and membrane-targetingfunctions (Lee et al Cell 1999). The sequence at the extreme C-terminusof PTEN is similar to sequences known to have binding affinity for PDZdomain-containing proteins. PTEN is a dual specificity phosphatase thatdisplays a pronounced preference for acidic substrates (Myers et al PNAS1997). Importantly, PTEN possesses lipid phosphatase activity,preferentially dephosphorylating phosphoinositides at the D3 position ofthe inositol ring. It is one of two enzymes known to dephosphorylate theD3 position in inositol phospholipids.

[0007] Since solid tumor progression is dependent on the induction ofangiogenic signals and augmented angiogenesis contributes to the highmortality associated with many cancers, there is a need to elucidate thecellular components that participate in these processes. The urgency ofsuch investigations is underscored by the fatal nature of highlymalignant brain tumors and the fact that the degree of tumorinvasiveness is directly correlated with enhanced angiogenesis.Furthermore, elucidation of cellular components that contribute to theangiogenic switch facilitates the identification of therapeutic agentsand delivery methods useful for the treatment of such malignantdiseases.

[0008] PTEN phosphatase activity has also been implicated in manycellular biochemical reactions. It is an object of the invention to alsoprovide methods for the identification of agents which impact PTENmodulation of immunoreceptors, AKT, PI3 kinase and p53 signaling.Methods of use of agents so identified are also within the scope of theinvention.

SUMMARY OF THE INVENTION

[0009] PTEN is a pivotal signaling molecule which modulates a widevariety of cellular processes. These cellular processes includeangiogenesis, cellular migration, immunoreceptor modulation, p53signaling and apoptotic cell death, PI3 and AKT signaling. Mutations inPTEN have been associated with the highly malignant progression of braintumors. A hallmark of this malignant progression is a dramatic increasein angiogenesis and invasiveness mediated by the concomitant formationof new blood vessels.

[0010] Thus, in accordance with the present invention methods for thetreatment of cancer associated with PTEN mutation are provided.Exemplary methods include delivery of a native PTEN encoding nucleicacid to cancer cells such that the native PTEN protein is expressed.Additional methods for the treatment of cancer in accordance with thepresent invention entail the administration of at least one agentselected from the group consisting of PTEN agonists, PI3 kinaseinhibitors and AKT inhibitors. The aforementioned treatment protocolsmay also comprise the administration of conventional chemotherapeuticagents.

[0011] In another aspect of the invention, methods for the prevention ofaberrant angiogenesis are also provided. Aberrant angiogenesis isassociated with several diseases. These include not only cancer, butautoimmune disease, arthritis, systemic lupus erthymatosis, inflammatorybowel disease, coronary artery disease, cerebrovascular disease, andatherosclerosis. Methods for the administration of at least one agentselected from the group consisting of native PTEN encoding nucleicacids, PTEN agonists, PI3 kinase inhibitors and AKT inhibitors for theinhibition or prevention of aberrant angiogenesis are also disclosedherein.

[0012] PTEN has also been implicated in immunoreceptor modulation. Thus,in yet another aspect of the invention, methods for inhibiting theimmune response in target cells are provided. PTEN agonists, PI3 kinaseinhibitors and/or AKT inhibitors are administered to patients to preventor inhibit immunoreceptor signaling. Such agents should have efficacy inthe treatment of graft rejection or graft versus host disease.

[0013] In yet another aspect of the invention, methods for regulatingp53 mediated gene expression are also provided. Such methods entail theadministration of native PTEN, PTEN agonists and/or PI3 kinaseinhibitors or AKT inhibitors to induce functional p53 in tumor cells.Such agents effectively increase chemosensitity and/or radiosensitivityof tumor cells by stimulating p53 mediated apoptotic cell death.

[0014] Given the widespread effects of PTEN, methods for identifyingagents which modulate PTEN activity are also provided. Exemplary assaysinclude those which assess alterations in activated AKT levels,alterations in microvessel formation, alterations in TSP1 levels,alterations in VEGF levels, alterations in TIMP3 levels, alterations inMMP9 activation and alterations PTEN phosphatase activity levels in thepresence and absence of such test agents.

[0015] Also provided in accordance with the present invention are highthroughput screening methods for identifying small molecules which haveaffinity for PTEN or fragments thereof. Small molecules so identifiedare within the scope of the present invention and may optionally befurther characterized in the functional assays described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIGS. 1A and 1B are a Western blot and a graph showing that stableexpression of PTEN and PTEN mutants in U87MG cells regulates AKT. (FIG.1A) Cell lysates from the U87MG (U87) cell line and U87 cells infectedwith a retroviral vector encoding PTEN (pBabe-Puro-PTEN) or mutants ofPTEN (pBabe-Puro-PTEN-G129E or R130M) were resolved by SDS-PAGE, equalamounts of proteins were loaded per lane and immunoblotted with antiserato PTEN, phospho-AKT and total AKT, and visualized by enhancedchemiluminescence. The basal levels of PTEN (top), phosphorylated AKT(ser 473) (middle) and total AKT (bottom) are shown. The status of thePTEN gene in each stable cell line was designated as: WT.E1 and WT.C7two separate clones expressing wild type PTEN. R130M and G129E aremutated PTEN proteins. R130M is inert as both a protein and a lipidphosphatase. The G129E PTEN can dephosphorylate acidic phosphopeptides,but cannot dephosphorylate the lipid substrate, PIP3. The U87MG (U87)cell line is the parental cell line isolated from a human glioblastomamultiforme patient. (FIG. 1B) Comparison of in vitro growth of U87MGcells transduced with mutants of PTEN. Equal number of cells (1×105)were incubated in RPMI +10% FBS for different times and cell numberswere quantitated by direct cell counting.

[0017]FIGS. 2A, 2B and 2C present data showing the effects of PTEN ongrowth of U87MG cells in vivo. (FIG. 2A) Cell growth in vivo. In orderto determine the rate of cell growth in vivo, equal amount of cells(5×10⁶) from each cell line were implanted at the right ventral flank bysubcutaneous injection (see legend). The formation and growth of thesubcutaneous tumor was monitored and the volume of the tumor wasdetermined by a three dimensional measurement at the times indicated(day 0, the date of implantation, no tumor is detected). Data wereanalyzed by Student's t-test and differences were significant comparingthe PTEN deficient (U87MG, R130M, G129E) to the wild type PTEN (WT.E1,WT.C7), n=5, number of mice p<0.0001 (FIG. 2B) Stereophotography ofsubcutaneous tumor sites in mice implanted with the parental U87 tumor,PTEN minus (left panel) versus wild type PTEN reconstituted tumor cells(right panel). These tumors represent 25 and 42 days after implantationfor PTEN minus versus wild type PTEN reconstituted tumors, respectively.(FIG. 2C) Immunoblot of cryostat tissue sections from subcutaneous tumorfor the expression pattern of PTEN, AKT and phosphorylated AKT. Frozentissue sections were solubilized in Laemmli sample buffer, total proteinwas quantitated and equal protein was loaded on SDS PAGE. The data shownare representative of tissue analysis from 5 animals per experimentalgroup.

[0018] FIGS. 3A-3E show that PTEN suppresses angiogenesis.Immunohistochemical analysis of staining with CD31 antibody to evaluatethe angiogenesis response within the parental U87MG tumor (FIG. 3A) andPTEN reconstituted tumors (FIG. 3B) implanted into the subcutaneoustissue. In PTEN minus and tumors expressing mutants of PTEN, there aremore new vessels formed (angiogenesis) (upper panel, arrow indicated)than in wild-type PTEN reconstituted tumor (lower panel), indicating thePTEN has direct influence on angiogenesis during tumor growth. (FIG. 3C)Microvessel density (MVD) counts were performed on tumor tissue stainedwith anti-CD31 antibody to determine effect of expression of PTEN andspecific PTEN mutants (G129E or R130M) on tumor induced angiogenesis.Bars represent standard deviation, 5 animals per group. Statisticalanalysis by Student's t-test demonstrate significant difference betweenMVD of PTEN null and PTEN catalytic mutants as compared to wild typePTEN reconstituted tumors, n=5, number of mice p<0.001. (FIG. 3D) PTENregulates the expression of thrombospondin-1 (TSP-1) in U87MG cells.RNAase protection assay was used to measure levels of TSP-1 mRNA in wildtype PTEN expressing U87 cells or cells transduced with a mutantcatalytically dead PTEN (G129R). U87MG cells were infected withretrovirus encoding wild type PTEN (WT), the catalytically dead, G129Rmutant (GR) or empty vector retrovirus (−) and selected for 10 days inpuromycin. RNA was harvested and RNAase protection assays were carriedout using probes for TSP-1 and GAPDH. A probe for glyceraldehydephosphate dehydrogenase (GAPDH) was used as a normalization control.(FIG. 3E) Thrombospondin immunoblot analysis. U87MG transduced with wildtype PTEN (WT) or a catalytic mutant PTEN (G129R) in an ecdysoneinducible expression system were induced (48 hours) with 0.5 μMmuristirone or assayed without induction to determine the effect of PTENexpression on the induction of TSP-1 by Western blotting. Supernatantsfrom cells were prepared and proteins resolved on SDS-PAGE and probedwith anti-TSP-1 antibody. There is clear up-regulation of TSP-1 in wildtype PTEN transduced U87 cultures compared to U87 cells expressing thelipid phosphatase deficient G129R mutant PTEN.

[0019]FIG. 4 is a Western blot showing the effect of constitutive PTENreconstitution on VEGF expression in U87MG cells. VEGF immunoblotanalysis of parental U87MG or PTEN wild type or mutant reconstitutedtumor cell lysates revealed a dramatic suppression of VEGF in wild typeand G129E mutant PTEN reconstituted tumor cells.

[0020] FIGS. 5A-E are a series of micrographs and a graph showing theeffects of PTEN reconstitution on survival in an orthotopic brain tumormodel. Equivalent number of parental U87 cells or U87 cellsreconstituted with wild type or mutant alleles of PTEN (seelegend)(1×106 cells) were implanted in right frontal lobe of nude mice.Stereophotography of whole brains from mice implanted with U87MG tumorcells (day 25) (FIG. 5A), or PTEN reconstituted (day 42) (FIG. 5B). Theimplantation site is shown by position of arrow in the wild type PTENreconstituted tumor, (FIG. 5C) A R130M PTEN reconstituted tumorimplanted into a nude mouse brain (magnification ×20). (FIG. 5D) A G129EPTEN reconstituted tumor implanted into a nude mouse brain. (FIG. 5E)Survival plots for mice implanted with PTEN minus or parental U87 cellstransduced with mutants of PTEN as shown. Survival data represents 15animals per experimental group. n=15, p<0.0001 for difference observedbetween the PTEN + and PTEN − groups for survival.

[0021]FIG. 6 is a blot showing that PTEN induces the expression ofTIMP-3. PTEN regulates the expression of tissue inhibitor ofmetalloproteinase (TIMP-3) in U87MG cells. RNAase protection assay wasused to measure levels of TIMP-3 mRNA in wild type PTEN expressing U87cells or cells transduced with a mutant catalytically dead PTEN (G129R).U87MG cells were infected with retrovirus encoding wild type PTEN (WT),the catalytically dead, G129R mutant (GR) or empty vector retrovirus (−)and selected for 10 days in puromycin. RNA was harvested and RNAaseprotection assays were carried out using probes for TIMP-3 and GAPDH. Aprobe for glyceraldehyde phosphate dehydrogenase (GAPDH) was used as anormalization control.

[0022]FIG. 7 is a gel showing that PTEN suppresses MMP-9 collagenolyticactivity in vivo. Reverse zymography was used to evaluate the effect ofPTEN reconstitution on collagenolytic activity within tumor tissue. Theenzymatic activities of MMP-2 and MMP-9 were detected based on molecularweight and Western blot analysis (data not shown).

[0023]FIG. 8 is a graph showing the effects of PTEN reconstitution ontumor invasion. Equivalent numbers of parental U87 cells or U87 cellsreconstituted with wild type or mutant forms PTEN. A transwell systemcoated with Matrigel (10 ug/ml) was used to assess the invasiveproperties of U87 cells versus PTEN reconstituted U87 cells in vitro.These data demonstrated that PTEN regulates the capacity of tumor cellsto invade a complex matrix barrier.

[0024]FIGS. 9A and 9B are a graph and a blot showing that dominantnegative Syk inhibits phagocytosis The phagocytosis of IgG sensitizedsRBCs by J774A.1 was measured in cells infected with empty vectorrecombinant vaccinia virus or virus containing dominant negative Syk(D/N Syk). The cells were infected with the respective viruses for 4 hat 37° C. with 5% CO₂, after which they were subjected to IgG sensitizedsRBCs in fresh medium at a target to effector ratio equal to 100:1 for 2h at 37° C. with 5% CO₂. Nonengulfed sRBCs were lysed by water shock andthe cells were fixed and stained with Wright-Giemsa staining beforecounting the phagocytic index. (FIG. 9A) Quantitation of phagocytosis ofIgG coated sRBCs by J774A.1 cells overexpressing D/N Syk. The columnsindicate phagocytic index of uninfected J774A.1 cells, cells infectedwith vaccinia virus containing vector only and cells infected withvaccinia virus containing D/N Syk. (FIG. 9B) J774A.1 cells infected withvaccinia virus containing D/N Syk expressed D/N Syk protein as shown inlane 3, while lane 1 represents untreated J774A.1 cells and lane 2represents J774A.1 cells infected with empty vector vaccinia virus as acontrol. The error bars represent standard deviation of the mean.

[0025]FIGS. 10A and 10B are graphs showing that Src and PI-3 kinase arerequired for ITAM signaling. Cells were treated with PP1, an inhibitorof Src family kinases, at concentrations of 10, 5 and 1 μM orwortmannin, an inhibitor of PI-3 kinase, at concentrations of 10, 5 and1 μg/ml along with an appropriate DMSO control for 1 h in DMEM with 10%FCS and then sensitized sRBCs were added at target to effector ratioequal to 100:1. (FIG. 10A) PP1 blocks the phagocytosis significantly at10 μM concentration and the effect is dose-dependent. (FIG. 10B)Wortmannin blocks phagocytosis significantly at 5 μg/ml. The columnsindicate phagocytic index of untreated J774A.1 cells treated with DMSO(control), PP1, or wortmannin. The error bars represent standarddeviation of mean.

[0026]FIGS. 11A and 11B are blots showing the effect of a dominantnegative Syk and Src inhibitor, PP1, on tyrosine phosphorylation of Cblin response to ITAM stimulation. FIG. 11A, upper panel shows the effectsof D/N Syk on tyrosine phosphorylation of Cbl in response to stimulationwith sensitized sRBCs. Lysates prepared from resting cells or cellsstimulated with sRBCs for 5 minutes were immunoprecipitated withpolyclonal anti-Cbl Ab and immunoblotted with anti-phosphotyrosineantibody. Lane 2 represents Cbl immunoprecipitated from resting J774A.1cells while lanes 5 and 8 represent Cbl immunoprecipitated from restingJ774A.1 cells infected with vaccinia virus containing plain vector ordominant negative Syk respectively. Lane 3 represents Cbl IP from cellsstimulated with sRBCs while lane 6 and 9 represents Cbl IP from cellsinfected with vaccinia virus containing plain vector or dominantnegative Syk respectively stimulated with sRBCs.(FIG. 11B, Upper panel)Cells were treated with PP1 to evaluate the role of the Src familykinases in Cbl tyrosine phosphorylation following stimulation withsensitized sRBCs. Lysates prepared from resting cells or cellsstimulated with sRBCs for 5 minutes were immunoprecipitated withpolyclonal anti-Cbl Ab and the resultant Cbl immunoprecipitates (Cbl IP)were immunoblotted with anti-phosphotyrosine antibody. Lane 2 representsCbl IP from resting J774A.1 cells while lane 5 represents Cbl IP fromresting J774A.1 cells treated with 10 μM PP1. Lane 3 represents Cbl IPfrom cells stimulated with sRBCs while lane 6 represents Cbl IP fromcells treated with PP1 and stimulated with sRBCs. (FIGS. 11A and 11B,lower panels) Anti-Cbl immunoblot of Cbl IP. Lysates prepared fromresting cells or cells stimulated with sRBCs for 5 minutes wereimmunoprecipitated with anti-Cbl antisera and immunoblotted withanti-Cbl antisera. Lanes are as designated in (A).

[0027]FIG. 12 is a graph showing that overexpression of PTEN in COS7cells inhibits ITAM signaling. Shows phagocytosis of IgG sensitizedsRBCs by COS cells transfected with FcγRIIa receptor, Syk, Cbl, PTEN ora trap mutant (C124S) of PTEN. The cells were transfected with episomalplasmids containing Syk, Cbl and/or PTEN for 12 h at 37° C. with 5% CO₂,after which were subjected to IgG sensitized sRBCs in fresh medium at atarget to effector ratio equal to 100:1 for two h at 37° C. with 5% CO₂.All experimental groups were transfected with FcRγIIA, Syk, and Cbl,data for Syk and Cbl, not shown. Nonengulfed sRBCs were lysed by watershock and the cells were fixed and stained with Wright-Giemsa stainingbefore counting the phagocytic index. A graphic representation of theinhibition of phagocytosis of IgG coated sRBCs by COS7 cellsoverexpressing PTEN is shown. The error bars represent standarddeviation of the mean. The red columns indicate the phagocytic index ofplain J774A.1 cells transfected with plasmid containing vector only orPTEN. The green bars represent effect of PTEN on the percent of cellsphagocytic for at least one SRBC.

[0028]FIG. 13 is a Western blot showing that PTEN regulates phospho-AKTlevels. A muristirone inducible expression system was used to expressPTEN or PTEN mutants in U87MG cells. Immunoblots of lysates derived fromU87 cells +/− induction for expression of wild type PTEN, wild typeHA-tagged PTEN, or G129R (GR) PTEN mutant of PTEN. were probed withantibodies specific for phospho-(S473)AKT or AKT.

[0029]FIG. 14 is a graph and a blot showing the effect of PTEN inductionon p53 transcription. U87MG cells expressing wild type PTEN, G129E orG129R mutants of PTEN under the control of muristirone (Western blot,insert) were transiently cotransfected with pRSVβ-galactosidase and mdm2luciferase. U87MG cells expressed similar levels of PTEN and G129R andslightly higher levels of G129E PTEN under control of a muristirone(+indicates muristirone added to cultures 24 hours prior to transfectionof reporter plasmids; lanes correspond to columns of bar graph).Induction of p53 dependent transcription was quantitated using βgalactosidase as an internal control for transfection efficiency.

[0030]FIG. 15 is a graph showing that PI-3 kinase inhibitors block tumorgrowth. Tumor volume was measured in DMSO treated mice or LY294002 (aPI3 kinase inhibitor) treated (100 mg/kg/day×2 weeks) mice; treatmentwas concomitant with tumor implantation.

[0031]FIG. 16 is a graph showing quantitation of CD31 positivemicrovessels within tumor tissue in the presence and absence ofLY294002. Note the dramatic inhibitory effect of LY294002 ontumor-induced angiogenesis. Bars represent standard deviation of themean (p<0.001).

[0032]FIG. 17 is a graph showing that administration of LY294002dramatically reduces the incidence of brain tumors.

[0033]FIG. 18 is a graph showing that the effect of LY294002 onchemosensitivity of glioma cells. −/−, no addition; −/1 mcM VP16, U87MGglioma cells exposed to 1 mcM VP16 only; −/5 mcM VP16, cells exposed to5 mcM VP16 only; LY/1 mcMVP16, cells exposed to 10 uM LY294002+1 mcMVP16; LY/5 mcM VP16, cells exposed to 10 uM LY294002+5 mcM VP16. Cellswere incubated for 48 hours with above components prior to MTT analysisfor viable cell numbers. Bars are standard deviation of meanobservation.

[0034]FIG. 19 is a graph showing the kinetic effect of LY294002 onEtoposide Chemosensitivity. −/−, no addition; LY294002/−, U87MG cellsexposed to 10 uM LY294002 alone; −/0.5 mcM VP16, cells exposed to 0.5mcM VP16 alone; −/1 mcM VP16, cells exposed to 1 mcM VP16 alone; −/5 mcMVP16, cells exposed to 5 mcM VP16 alone; LY/0.5 mcM VP16, cells exposedto 10 uM LY294002+0.5 mcM VP16; LY/1 mcM VP16, cells exposed to 10 uMLY294002+1 mcM VP16; LY/5 mcM VP16, cells exposed to 10 uM LY294002+5mcM VP16. Cells were assayed by MTT at different times after addition ofLY294002 and/or VP16 as shown.

[0035]FIGS. 20A and 20B depict a PTEN encoding nucleic acid (SEQ IDNO: 1) and the amino acid sequence (SEQ ID NO: 2) of PTEN, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Tumor progression, particularly in aggressive and malignanttumors, is associated with the induction of angiogenesis, a processtermed the angiogenic switch. Mutations of the tumor suppressor PTEN, aphosphatase with specificity for D3 phosphorylated inositolphospholipids, are associated with malignant and invasive tumorprogression. PTEN is, therefore, a critical regulator of tumorprogression that acts by modulating the angiogenic switch response.

[0037] To address the role of PTEN in the angiogenic switch response, acritical predictor of the metastatic potential of a tumor, a modelsystem was developed utilizing the U87MG glioma cell line. The U87MGcell line, which is null for PTEN, is a highly metastatic cell linederived from a human glioblastoma multiforme patient. U87MG glioma cellsstably reconstituted with PTEN cDNA were tested for growth in a nudemouse orthotopic brain tumor model. The introduction of wild type PTENinto U87MG cells results in decreased tumor growth in vivo and prolongedsurvival of mice implanted intracranially with these cells. PTENreconstitution diminished phosphorylation of AKT within thePTEN-reconstituted tumor, induced thrombospondin 1 expression, andsuppressed VEGF expression and angiogenic activity. These effects werenot observed in tumors reconstituted with the G129E mutant form of PTEN,in which lipid phosphatase activity is ablated. These data provide thefirst direct evidence that PTEN coordinately regulates the angiogenicswitch and the progression of gliomas to a malignant phenotype via theregulation of phosphoinositide-dependent signals which control p53transcriptional activity.

[0038] Thus in accordance with the present invention, methods areprovided for identifying and characterizing small molecules which impactPTEN modulated angiogenesis and tumor progression. A variety ofbiochemical assays are provided which will facilitate thecharacterization of such molecules. Exemplary assays include thosesuitable for assessing matrix degradation, angiogenesis, tumor invasionand suppression of matrix metalloproteinase 9 activity.

[0039] The regulatory role for PTEN phosphatase is widespread throughoutthe cell. As described in Example III, PTEN also regulates inflammatorysignaling. Immunoreceptor activation is associated with antibodydependent cell mediated toxicity, NK and CTL lysis of target cells, suchas tumor cells, parasitic cells and microorganisms. The data presentedherein indicate that PTEN controls immunoreceptor desensitization invivo. This observation provides the basis for the development of assaysand methods to identify and characterize small molecules which haveefficacy in the treatment immune disorders associated with hyperactiveinflammatory responses. Such molecules should also have efficacy in thetreatment of graft versus host disease and graft rejection. Methods ofuse of agents so identified are also in the scope of the invention. PTENinhibitors should effectively block immunoreceptor desensitizationthereby augmenting the immunotherapeutic activity of immune cells.Immune cells that may be targeted with these inhibitors include T cells,B cells, macrophages, dendritic cells, neutrophils, mast cells,eosinophils, and platelets.

[0040] A detailed analysis of the PTEN and PI-3 kinase signaling cascadeand its impact on p53 mediated transcription is provided in Example IV.In accordance with the present invention, it has been discovered thatp53 mediated transcription is dependent upon proper PTEN/PI3 kinasesignaling. These data also indicate that PI-3 kinase inhibitors have invivo antiangiogenic activity. It has also been discovered that PTENexerts control over p53 levels in cells as cells that contain mutatedPTEN have a marked reduction in functional p53 levels. Reduced p53function is associated with reduced sensitivity to stress orchemotherapy induced apoptosis. Thus, this data provides the basis forthe development of additional biological assays for assessing theeffects of small molecules which inhibit PTEN/PI3 kinase/p53 signaling.Such small molecules should also have efficacy in the treatment ofcancer.

[0041] PTEN activity has also been implicated in chemo- andradio-sensitivity as set forth in Example V. Thus, based on the datapresented herein, it has been discovered that activation of PTEN and thePI3 kinase pathway sensitizes cells to p53 mediated cell death throughthe control of p53 induced apoptosis. These observations thus providethe basis for additional methods for identifying efficaciouschemotherapeutic combination therapies which should be effective in thetreatment of cancer.

[0042] The following description sets forth the general proceduresinvolved in practicing the present invention. To the extent thatspecific materials are mentioned, it is merely for purposes ofillustration and is not intended to limit the invention. Unlessotherwise specified, general cloning and gene expression procedures,such as those set forth in Current Protocols in Molecular Biology,Ausubel et al. eds., JW Wiley and Sons, NY (1998)are utilized.

I. Definitions

[0043] Various terms relating to the biological molecules of the presentinvention are used hereinabove and also throughout the specificationsand claims.

[0044] “Nucleic acid” or a “nucleic acid molecule” as used herein refersto any DNA or RNA molecule, either single or double stranded and, ifsingle stranded, the molecule of its complementary sequence in eitherlinear or circular form. In discussing nucleic acid molecules, asequence or structure of a particular nucleic acid molecule may bedescribed herein according to the normal convention of providing thesequence in the 5′ to 3′ direction. With reference to nucleic acids ofthe invention, the term “isolated nucleic acid” is sometimes used. Thisterm, when applied to DNA, refers to a DNA molecule that is separatedfrom sequences with which it is immediately contiguous in the naturallyoccurring genome of the organism in which it originated. For example, an“isolated nucleic acid” may comprise a DNA molecule inserted into avector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryotic or eukaryotic cell or host organism.

[0045] When applied to RNA, the term “isolated nucleic acid” refersprimarily to an RNA molecule encoded by an isolated DNA molecule asdefined above. Alternatively, the term may refer to an RNA molecule thathas been sufficiently separated from other nucleic acids with which itwould be associated in its natural state (i.e., in cells or tissues). Anisolated nucleic acid (either DNA or RNA) may further represent amolecule produced directly by biological or synthetic means andseparated from other components present during its production.

[0046] “Natural allelic variants”, “mutants” and “derivatives” ofparticular sequences of nucleic acids refer to nucleic acid sequencesthat are closely related to a particular sequence but which may possess,either naturally or by design, changes in sequence or structure. Byclosely related, it is meant that at least about 75%, but often, morethan 90%, of the nucleotides of the sequence match over the definedlength of the nucleic acid sequence referred to using a specific SEQ IDNO. Changes or differences in nucleotide sequence between closelyrelated nucleic acid sequences may represent nucleotide changes in thesequence that arise during the course of normal replication orduplication in nature of the particular nucleic acid sequence. Otherchanges may be specifically designed and introduced into the sequencefor specific purposes, such as to change an amino acid codon or sequencein a regulatory region of the nucleic acid. Such specific changes may bemade in vitro using a variety of mutagenesis techniques or produced in ahost organism placed under particular selection conditions that induceor select for the changes. Such sequence variants generated specificallymay be referred to as “mutants” or “derivatives” of the originalsequence.

[0047] The present invention also includes methods of use for activeportions, fragments, derivatives and functional or non-functionalmimetics of PTEN polypeptides or proteins of the invention. An “activeportion” of PTEN polypeptide means a peptide that is less than the fulllength PTEN polypeptide, but which retains measurable biologicalactivity.

[0048] A “fragment” or “portion” of the PTEN polypeptide means a stretchof amino acid residues of at least about five to seven contiguous aminoacids, often at least about seven to nine contiguous amino acids,typically at least about nine to thirteen contiguous amino acids and,most preferably, at least about twenty to thirty or more contiguousamino acids. Fragments of the PTEN polypeptide sequence, antigenicdeterminants, or epitopes are useful for eliciting immune responses to aportion of the PTEN amino acid sequence.

[0049] A “derivative” of the PTEN polypeptide or a fragment thereofmeans a polypeptide modified by varying the amino acid sequence of theprotein, e.g. by manipulation of the nucleic acid encoding the proteinor by altering the protein itself. Such derivatives of the natural aminoacid sequence may involve insertion, addition, deletion or substitutionof one or more amino acids, and may or may not alter the essentialactivity of the original PTEN polypeptide.

[0050] As mentioned above, the PTEN polypeptide or protein of theinvention includes any analogue, fragment, derivative or mutant which isderived from a PTEN polypeptide and which retains at least one propertyor other characteristic of the PTEN polypeptide. Different “variants” ofthe PTEN polypeptide exist in nature. These variants may be allelescharacterized by differences in the nucleotide sequences of the genecoding for the protein, or may involve different RNA processing orpost-translational modifications. The skilled person can producevariants having single or multiple amino acid substitutions, deletions,additions or replacements. These variants may include inter alia: (a)variants in which one or more amino acids residues are substituted withconservative or non-conservative amino acids, (b) variants in which oneor more amino acids are added to the PTEN polypeptide, (c) variants inwhich one or more amino acids include a substituent group, and (d)variants in which the PTEN polypeptide is fused with another peptide orpolypeptide such as a fusion partner, a protein tag or other chemicalmoiety, that may confer useful properties to the PTEN polypeptide, suchas, for example, an epitope for an antibody, a polyhistidine sequence, abiotin moiety and the like. Other PTEN polypeptides of the inventioninclude variants in which amino acid residues from one species aresubstituted for the corresponding residues in another species, either atthe conserved or non-conserved positions. In another embodiment, aminoacid residues at non-conserved positions are substituted withconservative or non-conservative residues. The techniques for obtainingthese variants, including genetic (suppressions, deletions, mutations,etc.), chemical, and enzymatic techniques are known to the person havingordinary skill in the art.

[0051] To the extent such allelic variations, analogues, fragments,derivatives, mutants, and modifications, including alternative nucleicacid processing forms and alternative post-translational modificationforms result in derivatives of the PTEN polypeptide that retain any ofthe biological properties of the PTEN polypeptide, they are includedwithin the scope of this invention.

[0052] The term “functional” as used herein implies that the nucleic oramino acid sequence is functional for the recited assay or purpose.

[0053] The phrase “consisting essentially of” when referring to aparticular nucleotide or amino acid means a sequence having theproperties of a given SEQ ID No:. For example, when used in reference toan amino acid sequence, the phrase includes the sequence per se andmolecular modifications that would not affect the basic and novelcharacteristics of the sequence.

[0054] A “replicon” is any genetic element, for example, a plasmid,cosmid, bacmid, phage or virus, that is capable of replication largelyunder its own control. A replicon may be either RNA or DNA and may besingle or double stranded.

[0055] A “vector” is a replicon, such as a plasmid, cosmid, bacmid,phage or virus, to which another genetic sequence or element (either DNAor RNA) may be attached so as to bring about the replication of theattached sequence or element.

[0056] An “expression operon” refers to a nucleic acid segment that maypossess transcriptional and translational control sequences, such aspromoters, enhancers, translational start signals (e.g., ATG or AUGcodons), polyadenylation signals, terminators, and the like, and whichfacilitate the expression of a polypeptide coding sequence in a hostcell or organism.

[0057] The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 15-25 or morenucleotides, although it may contain fewer nucleotides. The probesherein are selected to be “substantially” complementary to differentstrands of a particular target nucleic acid sequence. This means thatthe probes must be sufficiently complementary so as to be able to“specifically hybridize” or anneal with their respective target strandsunder a set of pre-determined conditions. Therefore, the probe sequenceneed not reflect the exact complementary sequence of the target. Forexample, a non-complementary nucleotide fragment may be attached to the5′ or 3′ end of the probe, with the remainder of the probe sequencebeing complementary to the target strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprobe, provided that the probe sequence has sufficient complementaritywith the sequence of the target nucleic acid to anneal therewithspecfically.

[0058] The term “specifically hybridize” refers to the associationbetween two single-stranded nucleic acid molecules of sufficientlycomplementary sequence to permit such hybridization under pre-determinedconditions generally used in the art (sometimes termed “substantiallycomplementary”). In particular, the term refers to hybridization of anoligonucleotide with a substantially complementary sequence containedwithin a single-stranded DNA or RNA molecule of the invention, to thesubstantial exclusion of hybridization of the oligonucleotide withsingle-stranded nucleic acids of non-complementary sequence.

[0059] Amino acid residues described herein are preferred to be in the“L” isomeric form. However, residues in the “D” isomeric form may besubstituted for any L-amino acid residue, provided the desiredproperties of the polypeptide are retained.

[0060] All amino-acid residue sequences represented herein conform tothe conventional left-to-right amino-terminus to carboxy-terminusorientation.

[0061] The term “isolated protein” or “isolated and purified protein” issometimes used herein. This term refers primarily to a protein producedby expression of an isolated nucleic acid molecule of the invention.Alternatively, this term may refer to a protein that has beensufficiently separated from other proteins with which it would naturallybe associated, so as to exist in “substantially pure” form. “Isolated”is not meant to exclude artificial or synthetic mixtures with othercompounds or materials, or the presence of impurities that do notinterfere with the fundamental activity, and that may be present, forexample, due to incomplete purification, addition of stabilizers, orcompounding into, for example, immunogenic preparations orpharmaceutically acceptable preparations.

[0062] The term “substantially pure” refers to a preparation comprisingat least 50-60% by weight of a given material (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-95% by weightof the given compound. Purity is measured by methods appropriate for thegiven compound (e.g. chromatographic methods, agarose or polyacrylamidegel electrophoresis, HPLC analysis, and the like).

[0063] The term “tag,” “tag sequence” or “protein tag” refers to achemical moiety, either a nucleotide, oligonucleotide, polynucleotide oran amino acid, peptide or protein or other chemical, that when added toanother sequence, provides additional utility or confers usefulproperties, particularly in the detection or isolation, to thatsequence. Thus, for example, a homopolymer nucleic acid sequence or anucleic acid sequence complementary to a capture oligonucleotide may beadded to a primer or probe sequence to facilitate the subsequentisolation of an extension product or hybridized product. In the case ofprotein tags, histidine residues (e.g., 4 to 8 consecutive histidineresidues) may be added to either the amino- or carboxy-terminus of aprotein to facilitate protein isolation by chelating metalchromatography. Alternatively, amino acid sequences, peptides, proteinsor fusion partners representing epitopes or binding determinantsreactive with specific antibody molecules or other molecules (e.g., flagepitope, c-myc epitope, transmembrane epitope of the influenza A virushemaglutinin protein, protein A, cellulose binding domain, calmodulinbinding protein, maltose binding protein, chitin binding domain,glutathione S-transferase, and the like) may be added to proteins tofacilitate protein isolation by procedures such as affinity orimmunoaffinity chromatography. Chemical tag moieties include suchmolecules as biotin, which may be added to either nucleic acids orproteins and facilitate isolation or detection by interaction withavidin reagents, and the like. Numerous other tag moieties are known to,and can be envisioned by, the trained artisan, and are contemplated tobe within the scope of this definition.

[0064] As used herein, the terms “reporter,” “reporter system”,“reporter gene,” or “reporter gene product” shall mean an operativegenetic system in which a nucleic acid comprises a gene that encodes aproduct that when expressed produces a reporter signal that is a readilymeasurable, e.g., by biological assay, immunoassay, radioimmunoassay, orby calorimetric, fluorogenic, chemiluminescent or other methods. Thenucleic acid may be either RNA or DNA, linear or circular, single ordouble stranded, antisense or sense polarity, and is operatively linkedto the necessary control elements for the expression of the reportergene product. The required control elements will vary according to thenature of the reporter system and whether the reporter gene is in theform of DNA or RNA, but may include, but not be limited to, suchelements as promoters, enhancers, translational control sequences, polyA addition signals, transcriptional termination signals and the like.

[0065] The terms “transform”, “transfect”, “transduce”, shall refer toany method or means by which a nucleic acid is introduced into a cell orhost organism and may be used interchangeably to convey the samemeaning. Such methods include, but are not limited to, transfection,electroporation, microinjection, PEG-fusion and the like. The introducednucleic acid may or may not be integrated (covalently linked) intonucleic acid of the recipient cell or organism. In bacterial, yeast,plant and mammalian cells, for example, the introduced nucleic acid maybe maintained as an episomal element or independent replicon such as aplasmid. Alternatively, the introduced nucleic acid may becomeintegrated into the nucleic acid of the recipient cell or organism andbe stably maintained in that cell or organism and further passed on orinherited to progeny cells or organisms of the recipient cell ororganism. In other manners, the introduced nucleic acid may exist in therecipient cell or host organism only transiently.

[0066] A “clone” or “clonal cell population” is a population of cellsderived from a single cell or common ancestor by mitosis.

[0067] A “cell line” is a clone of a primary cell or cell populationthat is capable of stable growth in vitro for many generations.

[0068] An “antibody” or “antibody molecule” is any immunoglobulin,including antibodies and fragments thereof, that binds to a specificantigen. The term includes polyclonal, monoclonal, chimeric, andbispecific antibodies. As used herein, antibody or antibody moleculecontemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule such asthose portions known in the art as Fab, Fab′, F(ab′)2 and F(v).

[0069] II. Preparation of PTEN-Encoding Nucleic Acid Molecules, PTENProteins and Antibodies Thereto

[0070] The PTEN protein comprises, from amino- to carboxy-terminus, aprotein tyrosine phosphatase catalytic domain that has considerablehomology to the cytoskeletal protein tensin, a C2 domain that conferslipid-binding and membrane-targeting, and a PDZ domain-binding site thatcontributes to membrane localization and protein stability (Lee et alCell 1999, Wu et al PNAS 2000;). The amino-terminal catalytic domainincludes the HC(X)₅R sequence, which is the signature motif of proteintyrosine phosphatases. The Genbank accession number for the human PTENencoding nucleic acid molecule is NM000313. The amino acid sequence ofPTEN is provided in FIG. 20B. These sequences are also referred toherein as SEQ ID NO: 1 and SEQ ID NO: 2.

[0071] The PTEN protein, hereafter termed PTEN, is classified as a dualspecificity phosphatase, whose substrate targets include phosphorylatedproteins and inositol phospholipids. PTEN is distinguished by the factthat, unlike other dual specificity phosphatases, it preferentiallydephosphorylates phosphoinositides at the D3 position of the inositolring (Maehama et al Trends Cell Biol. 1999, Maehama et al J Biol Chem1998). PTEN is the product of the tumor suppressor gene PTEN/MMAC,mutations in which have been correlated with a number of different tumortypes, including those of the brain, prostate, endometrium, breast, andlung.

[0072] A. Nucleic Acid Molecules

[0073] Nucleic acid molecules encoding PTEN may be prepared by twogeneral methods: (1) They may be synthesized from appropriate nucleotidetriphosphates, or (2) they may be isolated from biological sources. Bothmethods utilize protocols well known in the art.

[0074] The availability of nucleotide sequence information, such as thefull length cDNA having Sequence I.D. No. 1, enables preparation of anisolated nucleic acid molecule of the invention by oligonucleotidesynthesis. Synthetic oligonucleotides may be prepared by thephosphoramadite method employed in the Applied Biosystems 38A DNASynthesizer or similar devices. The resultant construct may be purifiedaccording to methods known in the art, such as high performance liquidchromatography (HPLC). Long, double-stranded polynucleotides, such as aDNA molecule of the present invention, must be synthesized in stages,due to the size limitations inherent in current oligonucleotidesynthetic methods. Thus, for example, a large double-stranded DNAmolecule may be synthesized as several smaller segments of appropriatecomplementarity. Complementary segments thus produced may be annealedsuch that each segment possesses appropriate cohesive termini forattachment of an adjacent segment. Adjacent segments may be ligated byannealing cohesive termini in the presence of DNA ligase to constructthe entire protein encoding sequence. A synthetic DNA molecule soconstructed may then be cloned and amplified in an appropriate vector.

[0075] Nucleic acid sequences encoding PTEN may be isolated fromappropriate biological sources using methods known in the art. In apreferred embodiment, a cDNA clone is isolated from an expressionlibrary of human origin. In an alternative embodiment, genomic clonesencoding PTEN may be isolated.

[0076] Nucleic acids of the present invention may be maintained as DNAin any convenient cloning vector. In a preferred embodiment, clones aremaintained in plasmid cloning/expression vector, such as pBluescript(Stratagene, La Jolla, Calif.), which is propagated in a suitable E.coli host cell.

[0077] PTEN-encoding nucleic acid molecules of the invention includecDNA, genomic DNA, RNA, and fragments thereof which may be single- ordouble-stranded. Thus, this invention provides oligonucleotides (senseor antisense strands of DNA or RNA) having sequences capable ofhybridizing with at least one sequence of a nucleic acid molecule of thepresent invention, such as selected segments of the cDNA having SequenceI.D. No. 1. Such oligonucleotides are useful as agents to inhibit oraugment PTEN activity in cells or tissues. In particular, the presentinvention describes the use of PTEN encoding nucleic acids forreconstitution of PTEN activity in malignant cells or tissues for thepurposes anti-cancer therapy.

[0078] B. Proteins

[0079] Full-length PTEN of the present invention may be prepared in avariety of ways, according to known methods. The protein may be purifiedfrom appropriate sources, e.g., human or animal cultured cells ortissues, by immunoaffinity purification. However, this is not apreferred method due to the small amounts of protein likely to bepresent in a given cell type at any time. The availability of nucleicacids molecules encoding PTEN enables production of the protein using invitro expression methods known in the art. For example, a cDNA or genemay be cloned into an appropriate in vitro transcription vector, such apSP64 or pSP65 for in vitro transcription, followed by cell-freetranslation in a suitable cell-free translation system, such as wheatgerm or rabbit reticulocytes. In vitro transcription and translationsystems are commercially available, e.g., from Promega Biotech, Madison,Wis. or BRL, Rockville, Md.

[0080] Alternatively, according to a preferred embodiment, largerquantities of PTEN may be produced by expression in a suitableprocaryotic or eucaryotic system. For example, part or all of a DNAmolecule, such as the cDNA having Sequence I.D. No. 1, may be insertedinto a plasmid vector adapted for expression in a bacterial cell, suchas E. coli, or into a baculovirus vector for expression in an insectcell. Such vectors comprise the regulatory elements necessary forexpression of the DNA in the bacterial host cell, positioned in such amanner as to permit expression of the DNA in the host cell. Suchregulatory elements required for expression include promoter sequences,transcription initiation sequences and, optionally, enhancer sequences.

[0081] The PTEN produced by gene expression in a recombinant procaryoticor eucaryotic system may be purified according to methods known in theart. In a preferred embodiment, a commercially availableexpression/secretion system can be used, whereby the recombinant proteinis expressed and thereafter secreted from the host cell, to be easilypurified from the surrounding medium. If expression/secretion vectorsare not used, an alternative approach involves purifying the recombinantprotein by affinity separation, such as by immunological interactionwith antibodies that bind specifically to the recombinant protein. Suchmethods are commonly used by skilled practitioners.

[0082] The PTEN proteins of the invention, prepared by theaforementioned methods, may be analyzed according to standardprocedures. For example, such proteins may be subjected to amino acidsequence analysis, according to known methods.

[0083] The present invention also provides methods of use of antibodiescapable of immunospecifically binding to proteins of the invention.Polyclonal antibodies directed toward PTEN or fragments thereof may beprepared according to standard methods. In a preferred embodiment,monoclonal antibodies are prepared, which react immunospecifically withvarious epitopes of PTEN. Monoclonal antibodies may be preparedaccording to general methods of Köhler and Milstein, following standardprotocols. Polyclonal or monoclonal antibodies that immunospecificallyinteract with PTEN can be utilized for identifying and purifying suchproteins. For example, antibodies may be utilized for affinityseparation of proteins with which they immunospecifically interact.Antibodies may also be used to immunoprecipitate proteins from a samplecontaining a mixture of proteins and other biological molecules. Otheruses of anti-PTEN antibodies are described below.

[0084] III. Uses of PTEN-Encoding Nucleic Acids, PTEN Proteins andAntibodies Thereto

[0085] Tumor suppressor proteins constitute a functional family ofproteins known to be essential regulators of cellular proliferation and,as such, provide suitable targets for the development of therapeuticagents for modulating their activity in a cell. Since PTEN is a tumorsuppressor protein implicated in the etiology of many malignantdiseases, methods for identifying agents that modulate its activity areprovided. Agents so identified should have efficacy in the treatment ofa variety of malignant diseases. The use of PTEN modulating agents inconjunction with other known anti-cancer treatments such as chemotherapyand radiation therapy is also described. Moreover, such therapeuticagents will also be useful for modulating the activity of PTEN in othercellular systems. Since there are several diseases, other than cancer,in which abnormal angiogenesis contributes to the etiology of thedisease, the administration of PTEN modulating agents should providetherapeutic advantages for the treatment of these conditions.Utilization of therapeutic agents that modulate PTEN activity could alsobe used effectively to treat disorders characterized hyperactivity ofthe inflammatory immune response.

[0086] A. PTEN-Encoding Nucleic Acids

[0087] PTEN-encoding nucleic acids may be used for a variety of purposesin accordance with the present invention. PTEN-encoding DNA, RNA, orfragments thereof may be used as probes to detect the presence of and/orexpression of genes encoding PTEN. Methods in which PTEN-encodingnucleic acids may be utilized as probes for such assays include, but arenot limited to: (1) in situ hybridization; (2) Southern hybridization(3) northern hybridization; and (4) assorted amplification reactionssuch as polymerase chain reactions (PCR).

[0088] Nucleic acid molecules, or fragments thereof, encoding PTEN mayalso be utilized to control the expression of PTEN, thereby regulatingthe amount of protein available to participate in tumor suppressorsignaling pathways. Alterations in the physiological amount of PTEN mayact synergistically with chemotherapeutic agents used to treat cancer.In one embodiment, the nucleic acid molecules of the invention will beused to restore PTEN expression to normal cellular levels or overexpressPTEN in a population of malignant cells. In this embodiment,reconstitution of signaling events downstream of PTEN abrogates theaberrant cellular proliferation observed in malignant cells.

[0089] In another embodiment, the nucleic acid molecules of theinvention may be used to decrease expression of PTEN in a population oftarget cells. In this embodiment, oligonucleotides are targeted tospecific regions of PTEN-encoding genes that are critical for geneexpression. The use of antisense oligonucleotides to decrease expressionlevels of a pre-determined gene is known in the art. In a preferredembodiment, such antisense oligonucleotides are modified in various waysto increase their stability and membrane permeability, so as to maximizetheir effective delivery to target cells in vitro and in vivo. Suchmodifications include the preparation of phosphorothioate ormethylphosphonate derivatives, among many others, according toprocedures known in the art. The use of antisense oligonucleotides forthe modulation of PTEN expression is disclosed in U.S. Pat. No.6,020,199, filed Feb. 1, 2000, the entire disclosure of which isincorporated by reference.

[0090] As described above, PTEN-encoding nucleic acids are also used toadvantage to produce large quantities of substantially pure PTENprotein, or selected portions thereof. In a preferred embodiment, theN-terminal “catalytic domain” of PTEN is produced by expression of anucleic acid encoding the domain. The full-length protein or selecteddomain is thereafter used for various research, diagnostic andtherapeutic purposes, as described below.

[0091] B. PTEN Protein and Antibodies

[0092] Purified PTEN, or fragments thereof, may be used to producepolyclonal or monoclonal antibodies which also may serve as sensitivedetection reagents for the presence and accumulation of PTEN (orcomplexes containing PTEN) in cultured cells or tissues from livingpatients (the term “patients” refers to both humans and animals).Recombinant techniques enable expression of fusion proteins containingpart or all of the PTEN protein. The full length protein or fragments ofthe protein may be used to advantage to generate an array of monoclonalantibodies specific for various epitopes of the protein, therebyproviding even greater sensitivity for detection of the protein in cellsor tissue.

[0093] Polyclonal or monoclonal antibodies immunologically specific forPTEN may be used in a variety of assays designed to detect andquantitate the protein, which may be useful for diagnosing aPTEN-related malignant disease in a patient. Such assays include, butare not limited to: (1) flow cytometric analysis; (2) immunochemicallocalization in PTEN in cultured cells or tissues; and (3) immunoblotanalysis (e.g., dot blot, Western blot) of extracts from various cellsand tissues. Additionally, as described above, anti-PTEN can be used forpurification of PTEN (e.g., affinity column purification,immunoprecipitation).

[0094] Anti-PTEN antibodies may also be utilized as therapeutic agentsto block the normal functionality of PTEN in a target cell population,such as an inflammatory cell. Thus, similar to the antisenseoligonucleotides described above, anti-PTEN antibodies may be deliveredto a target cell population by methods known in the art (i.e. throughvarious lipophilic carriers that enable delivery of the compound ofinterest to the target cell cytoplasm) where the antibodies may interactwith intrinsic PTEN to render it nonfunctional.

[0095] From the foregoing discussion, it can be seen that PTEN-encodingnucleic acids and PTEN proteins of the invention can be used to modulatePTEN gene expression and protein activity for the purposes of assessingthe impact of PTEN modulation on the regulation of proliferativepathways of a cell or tissue sample. It is expected that these toolswill be particularly useful for the treatment of human neoplasticdisease in that PTEN-encoding nucleic acids, proteins and antibodies areexcellent candidates for use as therapeutic agents.

[0096] Although the compositions of the invention have been describedwith respect to human therapeutics, it will be apparent to one skilledin the art that these tools will also be useful in animal and culturedcell experimentation with respect to various malignancies and/or otherconditions manifested by alterations in cellular proliferation. Astherapeutics, they can be used either alone or as adjuncts to otherchemotherapeutic drugs to improve the effectiveness of such anti-canceragents.

III. Therapeutics A. Rational Drug Design

[0097] Since PTEN is a tumor suppressor protein implicated in theetiology of many malignant diseases, including, but not limited to,those of the brain, prostate, endometrium, and lung, methods foridentifying agents that modulate its activity should result in thegeneration of efficacious therapeutic agents for the treatment of avariety of malignant and inflammatory diseases.

[0098] The crystal structure of PTEN, solved in 1999, revealed that the403 amino acid protein comprises three domains of known function. Theseare the N terminal catalytic domain(residues 1-185), the C2 domain(residues 186-349) that participates in membrane binding and catalysisand the C terminal tail region (residues 350-403). See FIG. 20. Each ofthese domains provide suitable targets for the rational design oftherapeutic agents which modulate PTEN activity. Particularly preferredregions are the N terminal and C2 domains, specifically regionsincluding certain unique residues within and adjacent to the P loop, theWPD loop and the TI loop. It is these residues that participate inspecific PIP3 substrate recognition and catalysis thereof. Anothersuitable region includes the C terminal tail which participates in PTENregulatory and degradation in vivo. Small peptide moleculescorresponding to these regions may be used to advantage in the design oftherapeutic agents which effectively modulate the activity of PTEN, PI-3kinase cascades, AKT cascades, as well as p53-mediated transcription andcell death.

[0099] PTEN is phosphorylated on tyrosine, serine and threonineresidues. Agents which affect the phosphorylation state of the proteinwill also be screened as those small molecules which affectphosphorylation of PTEN should also modulate PTEN interactions withother proteins. The DLDLTYIYP motif (residues 22-30; SEQ ID NO: 3) atthe extreme N terminus of PTEN contains a YxxP motif (SEQ ID NO: 4), apossible docking site for adapter proteins like crk and crkl via SH2interactions. Another motif, YFSPN (SEQ ID NO: 5) in the C terminus hasbeen identified as the binding site for crk and crkl. The YLVLTL motif(SEEQ ID NO: 6) in the extreme C terminus is a site for SH2 interactionswith Shc or SHP-1. The YSYL motif (SEQ ID NO: 7), which contains atyrosine at position 178, is 100% conserved from Drosophila to man.Other tyrosine phosphorylated motifs include: YRNNIDD (SEQ ID NO: 8), Yat position 46, a sequence present in the catalytic domain identified asa binding site for Grb2 via its SH2 domain.

[0100] Binding and inhibition of PTEN phosphatase may be assessed usingrecombinant wild type PTEN or mutants of PTEN and appropriate PIP₃substrates to measure dephosphorylation of PIP₃ at D3 position. Agentswhich modulate PTEN phosphatase action should have efficacy in thetreatment of cancer and inflammatory diseases. The dephosphorylation ofphosphatidylinositol 3,4,5,-trisphosphate (PIP₃) is carried out in areaction mixture consisting of 100 mM Tris-HCl (pH 8), 10 mMdithiothreitol, 0.5 mM diC₁₆ phosphatidylserine (PS), 25 uM PIP₃, diC₁₆,BIOMOL PH-107 (BIOMOL, Inc.) and 50 μg/ml purified recombinant PTEN.Lipids were prepared in organic solvents and dispensed into 1.5 mlmicrofuge tubes followed by solvent removal under reduced pressure.Buffer is then added and a lipid suspension is formed by sonication.PTEN phosphatase assays are initiated by the addition of PTEN andcarried out at 37° C. At different time points, 15 μl of 100 μM NEM(N-ethylmaleimide) is added to 10 μl of reaction mixture followed byrapid centrifugation 18,000× g for 15 minutes at 4° C. Liberatedinorganic phosphate is detected in twenty microliters of supernatantusing the Malachite green assay and an inorganic phosphate standardcurve. Malachite green reaction with inorganic phosphate is detectedspectrophotometrically at 620 nm wavelength. The N terminal domain alsocontains the P-loop (HCKAGKGR, residues (123-130; SEQ ID NO: 9) which isunique to PTEN. Two basic residues, K125 and K128 at the active sitelikely account for the capacity of the P-loop to accommodate the largePIP₃ as a substrate. The cysteine residue at position 124 forms athiophosphate intermediate with the phosphorylated PIP₃ molecule. R130is involved in catalysis of this phosphoester linkage. The His atposition 123 and Glycine at position 127 are also critical for theconformation of the P-loop structure. The trough region defined by theactive site is extended to 8 angstroms in depth and 5×11 angstromopening to the active site. This site will be targeted for molecularmodeling to develop inhibitors specific for the PTEN phosphatase.Comparisons with other phosphatases (PTP and PTP1B) with PIP₃ activitywill facilitate identification of those agents which specificallyinteract with PTEN.

[0101] The WPD loop or DHNPPQ motif (residues 92-97; SEQ ID NO: 10) isequally important in catalysis in that mutation of Asp-92 results in aloss of catalytic activity. This aspartic acid residue acts as a generalacid to protonate the phenolic oxygen atom of a tyrosyl group fortyrosine phosphatases. These data suggest that the mechanistic action ofPTEN is similar to that of tyrosine phosphatases during hydrolysis ofthe phosphate ester in PIP₃.

[0102] The invariant sequence in the WPD loop will also be used in acombinatorial drug screen based on the electrostatic chargecharacteristics of this region of the PTEN molecule. Functional sidechains of these amino acids will be targeted with organic moleculeswhich mimic or disrupt the WPD interaction with the P-loop and TI-loopresidues. For these screens a PTEN inert organic scaffold will bedeveloped to allow for detection of organic molecules which specificallybind PTEN or PTEN fragments.

[0103] The structure of the TI loop in the N terminal domain facilitatesPTEN-mediated dephosphorylation of the PIP₃ molecule by providing anelongated and enlarged catalytic site. This is in contrast to other dualspecificity phosphatases. Residues (164-174) in the T1 loop includeKGVTIPSQRRY; (SEQ ID NO: 17). These residues are 100% conserved. Theserine residue at position 170, R at 173 and Y at 174 in this PTENpeptide are important in maintaining the interaction between the TI loopand the C2 domain and are often mutated in human tumors.

[0104] The TI loop of PTEN is in close proximity to the C2 domain andmaintains a rigid interface to promote the open configuration for PIP₃binding to PTEN. A 100% conserved region in C2 domain, HFWVNTFFI,(SEQ IDNO: 11) will also be used to screen a combinatorial library for organicmolecules which bind to this motif.

[0105] Small molecules which have affinity for the C terminal tail ofPTEN will also be screened and characterized. Molecules so identifiedshould decrease degradation of PTEN in cells by interfering withphosphorylation of residues, S380, T382 and T383 within the sequenceRYSDTTDS (SEQ ID NO:16) at the extreme C terminus. Screens will beperformed with recombinant GST PTEN protein comprising the last 50 aminoacids (residues 350-403) of PTEN to identify those agents which haveaffinity for this region of PTEN. This methodology should identifyagents that will antagonize the phosphorylation of PTEN tail and/orinterfere with PDZ binding thereby blocking PTEN interaction with theplasma membrane. Agents which disrupt this PDZ interaction will likelyinterfere with PTEN degradation and hence increase PTEN activity levelsin vivo.

[0106] Another suitable target present in the C-terminal domain of PTENis the PEST domain. This region encodes a site for ubiquitin mediateddegradation of PTEN which, if blocked, should augment PTEN activity bypreventing its degradation. Other sequences present in the C terminus ofPTEN between residues 251-351 include TLTKNDLD-FTKTV (SEQ ID NO:12),GDIKVEF-FTKTV (SEQ ID NO:13), DKANKDKAN-FTKTV (SEQ ID NO:14).

[0107] The present invention is not only directed to methods for therational design and screening of agents having binding affinity to theparticular peptide sequences described above. Several PTEN mutants aredisclosed herein which may also be used to advantage to identifymolecules which modulate PTEN activity. For example, the C124S mutant ofPTEN provides an ideal target for the initial screening of therapeuticagents given its increased affinity for substrates and substratetrapping capacity in vivo. Use of this mutant PTEN in the assays of theinvention should result in the identification and characterization ofphosphoinositol D3 analogs that inhibit PTEN activity. Such agentsshould include organic chemicals with the capacity to disrupt thevicinal sulfhydryl interaction of C124 with the phosphate group requiredto form a thiol-phosphate intermediate for cleavage of the inositolphosphate bond. Agents which so modulate action of the C124S mutant ofPTEN will then be further assessed in functional phosphatase assays.

[0108] Monoclonal antibodies, proteins, protein fragments, peptides andpeptidomimetic analogs of peptides which simulate the binding site forPIP₃ as well as structural homologs of phosphoinositides phosphorylatedin D3 position, or substituted in the D3 position with other negativelycharged functional groups, will be screened for capacity to bind andmodulate PTEN phosphatase activity in vitro. Molecular modeling shouldfacilitate the identification of specific organic molecules withcapacity to bind to the active site of PTEN based on conformation or keyamino acid residues required for catalytic function. A combinatorialchemistry approach will be used to identify molecules with greatestactivity and then iterations of these molecules will be developed forfurther cycles of screening.

[0109] The PTEN polypeptide or fragment employed in drug screeningassays may either be free in solution, affixed to a solid support orwithin a cell. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantpolynucleotides expressing the polypeptide or fragment, preferably incompetitive binding assays. Such cells, either in viable or fixed form,can be used for standard binding assays. One may determine, for example,formation of complexes between a PTEN polypeptide or fragment and theagent being tested, or examine the degree to which the formation of acomplex between a PTEN polypeptide or fragment and a known substrate isinterfered with by the agent being tested.

[0110] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to PTENpolypeptides and is described in detail in Geysen, PCT publishedapplication WO 84/03564, published on Sep. 13, 1984. Briefly stated,large numbers of different, small peptide test compounds, such as thosedescribed above, are synthesized on a solid substrate, such as plasticpins or some other surface. The peptide test compounds are reacted withPTEN polypeptide and washed. Bound PTEN polypeptide is then detected bymethods well known in the art.

[0111] A further technique for drug screening involves the use of hosteukaryotic cell lines or cells (such as described above) which have anonfunctional PTEN gene. These host cell lines or cells are defective atthe PTEN polypeptide level. The host cell lines or cells are grown inthe presence of drug compound. The rate of cellular proliferation andtransformation of the host cells is measured to determine if thecompound is capable of regulating the proliferation and transformationof PTEN defective cells.

[0112] Another approach entails the use of phage display librariesengineered to express fragment of PTEN on the phage surface. Suchlibraries are then contacted with a combinatorial chemical library underconditions wherein binding affinity between the PTEN peptide and thecomponents of the chemical library may be detected. U.S. Pat. Nos.6,057,098 and 5,965,456 provide methods and apparatus for performingsuch assays.

[0113] The goal of rational drug design is to produce structural analogsof biologically active polypeptides of interest or of small moleculeswith which they interact (e.g., agonists, antagonists, inhibitors) inorder to fashion drugs which are, for example, more active or stableforms of the polypeptide, or which, e.g., enhance or interfere with thefunction of a polypeptide in vivo. See, e.g., Hodgson, (1991)Bio/Technology 9:19-21. In one approach, discussed above, thethree-dimensional structure of a protein of interest or, for example, ofthe protein-substrate complex, is solved by x-ray crystallography, bynuclear magnetic resonance, by computer modeling or most typically, by acombination of approaches. Less often, useful information regarding thestructure of a polypeptide may be gained by modeling based on thestructure of homologous proteins. An example of rational drug design isthe development of HIV protease inhibitors (Erickson et al., (1990)Science 249:527-533). In addition, peptides (e.g., PTEN polypeptide) maybe analyzed by an alanine scan (Wells, (1991) Meth. Enzym. 202:390-411).In this technique, an amino acid residue is replaced by Ala, and itseffect on the peptide's activity is determined. Each of the amino acidresidues of the peptide is analyzed in this manner to determine theimportant regions of the peptide. It is also possible to isolate atarget-specific antibody, selected by a functional assay, and then tosolve its crystal structure. In principle, this approach yields apharmacore upon which subsequent drug design can be based.

[0114] It is possible to bypass protein crystallography altogether bygenerating anti-idiotypic antibodies (anti-ids) to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of the anti-ids would be expected to be an analog ofthe original molecule. The anti-id could then be used to identify andisolate peptides from banks of chemically or biologically produced banksof peptides. Selected peptides would then act as the pharmacore.

[0115] Thus, one may design drugs which have, e.g., improved PTENpolypeptide activity or stability or which act as inhibitors, agonists,antagonists, etc. of PTEN polypeptide activity. By virtue of theavailability of cloned PTEN sequences, sufficient amounts of the PTENpolypeptide may be made available to perform such analytical studies asx-ray crystallography. In addition, the knowledge of the PTEN proteinsequence provided herein will guide those employing computer modelingtechniques in place of, or in addition to x-ray crystallography.

[0116] Suitable peptide targets for identifying specific PTEN bindingand modulating agents are provided in Table I TABLE I PTEN peptidemotifs used to screen for PTEN agonists and inhibitors Phosphorylationsite motifs: number amino acid residue DLDLTYIYP  (22-30) SEQ ID NO: 3YLVLTL  (27-30) SEQ ID NO: 6 YRNNIDD  (46-52) SEQ ID NO: 8KGVTIPSQRRYVYYYSYLL (164-182) SEQ ID NO: 15 YSYL (178-181) SEQ ID NO: 7YFSPN (336-339) SEQ ID NO: 5 RYSDTTDS (378-385) SEQ ID NO: 16 CatalyticDomain motifs (1-185) HCKAGKR (P-loop) (123-130) SEQ ID NO: 9 DHNPPQ(WPD-loop)  (92-97) SEQ ID NO: 10 KGVTIPSQRRY (TI-loop) (164-174) SEQ IDNO: 17 C2 domain motifs (186-350) HFWVNTFFI (272-280) SEQ ID NO: 11 Cterminal tail related regions of interest (351-403) TLTKNDLD---FTKTV(PEST domain sequences) (319-351) SEQ ID NO: 12 GDIKVEF----FTKTV (PESTdomain sequences) (251-351) SEQ ID NO: 13 DKANKDKAN---FTKTV (PEST)(331-351) SEQ ID NO: 14 RYSDTTDS (pre-PDZ region) (378-385) SEQ ID NO:16 HTQITKV (PDZ-MAGI-2 interaction domain) (399-403) SEQ ID NO: 18

[0117] B. Pharmaceuticals and Peptide Therapies

[0118] The elucidation of the role played by PTEN in cellulartransformation and angiogenesis facilitates the development ofpharmaceutical compositions useful for treatment and diagnosis of PTENassociated disorders. These compositions may comprise, in addition toone of the above substances, a pharmaceutically acceptable excipient,carrier, buffer, stabilizer or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material may depend on the route ofadministration, e.g. oral, intravenous, cutaneous or subcutaneous,nasal, intramuscular, intraperitoneal routes.

[0119] Whether it is a polypeptide, antibody, peptide, nucleic acidmolecule, small molecule or other pharmaceutically useful compoundaccording to the present invention that is to be given to an individual,administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual.

C. Methods of Gene Therapy

[0120] As a further alternative, the nucleic acid encoding the authenticbiologically active PTEN polypeptide could be used in a method of genetherapy, to treat a patient who is unable to synthesize the active“normal” polypeptide or unable to synthesize it at the normal level,thereby providing the effect elicited by wild-type PTEN and suppressingthe occurrence of “abnormal” PTEN associated diseases such as cancer.

[0121] Vectors, such as viral vectors have been used in the prior art tointroduce genes into a wide variety of different target cells. Typicallythe vectors are exposed to the target cells so that transformation cantake place in a sufficient proportion of the cells to provide a usefultherapeutic or prophylactic effect from the expression of the desiredpolypeptide. The transfected nucleic acid may be permanentlyincorporated into the genome of each of the targeted cells, providinglong lasting effect, or alternatively the treatment may have to berepeated periodically.

[0122] A variety of vectors, both viral vectors and plasmid vectors areknown in the art, see U.S. Pat. No. 5,252,479 and WO 93/07282. Inparticular, a number of viruses have been used as gene transfer vectors,including papovaviruses, such as SV40, vaccinia virus, herpes virusesincluding HSV and EBV, and retroviruses. Many gene therapy protocols inthe prior art have employed disabled murine retroviruses.

[0123] Gene transfer techniques which selectively target the PTENnucleic acid to malignant tissues are preferred. Examples of thisinclude receptor-mediated gene transfer, in which the nucleic acid islinked to a protein ligand via polylysine, with the ligand beingspecific for a receptor present on the surface of the target cells.Microcapsule based delivery systems are also available for delivery ofnucleic acids to targeted cell types.

[0124] The following Examples are provided to describe the invention infurther detail. The Examples are intended to illustrate and not to limitthe invention.

EXAMPLE 1 Orthotopic Brain Tumor Model

[0125] To determine whether PTEN exerts control over angiogenesis and/orthe growth of glial tumors, an orthotopic brain tumor model wasdeveloped in which PTEN-deficient tumor cells were geneticallymanipulated in vitro and then stereotactically injected into the frontalcerebral cortex of nude mice. The U87MG cell line employed in thesestudies is derived from a patient diagnosed with glioblastomamultiforme, a highly malignant and uniformly fatal brain tumor. Thistumor and other human glioblastomas and glioblastoma cell lines containa mutation in both PTEN alleles (U87MG cells have a homozygous mutationin PTEN resulting in a null genotype).

[0126] In the orthotopic brain tumor model, 100% of mice implantedintracranially with the parental U87 cells display a highly invasive andangiogenic pattern of brain tumor growth that results in mortalitywithin 25-27 days. In view of these observations, the effect ofreconstituting expression of the PTEN gene in the parental U87MG (U87)cell line was explored.

[0127] Stable derivatives of the parental U87 cells were generatedfollowing transduction with retroviruses encoding cDNA for wild typePTEN or specific mutants of this phosphatase. In particular, missensemutations in the PTP signature motif were introduced to ascertain theimportance of the enzymatic activity of PTEN to its tumor suppressorfunction. Missense mutations included the G129E mutant, which displays aseverely attenuated ability to dephosphorylate inositol phospholipids,but retains normal enzymatic activity for phosphoproteins. Thebiological significance of the G129E mutant was underscored by the factthat its presence has been correlated with Cowden's disease andendometrial cancer. In another missense mutation generated, the R130Mmutant, all phosphatase activity has been abrogated (Myers et al PNAS1997, Funari et al PNAS 1997).

[0128] Tumor cells were characterized biochemically for levels ofactivated AKT (phospho-S473-AKT), growth in vitro and PTEN expression(FIG. 1). Anti-PTEN blots confirmed that parental U87 cells did notexpress PTEN and that following reconstitution of PTEN expression, U87cells expressed significant and comparable amounts of the wild type ormutant phosphatase protein. Expression of wild type PTEN, at levelssimilar to those observed in a mouse brain lysate, suppressed theactivated state of AKT observed in PTEN-deficient U87 cells (FIG. 1A,lanes 3 & 5). Following expression of the R130M and G129E mutant formsof PTEN, the levels of phospho-AKT were similar to those observed in theparental U87 cells (FIG. 1A, lanes 1, 2 & 4), suggesting that the lipidphosphatase activity of PTEN was essential for the effects on thePIP3-dependent activation of AKT. Interestingly, the growth of thedifferent PTEN-expressing U87 cell lines in vitro was similar in 2, 5and 10% fetal bovine serum (data not shown and FIG. 1B). Therefore, wecompared these cell lines further in our in vivo models.

[0129] Athymic nude mice were implanted subcutaneously and byintracranial injection. Production of subcutaneous tumors facilitatesmonitoring of tumor size and performance of direct biochemical analysisof tumor tissue for the examination PTEN expression and levels of AKTactivation without significant contamination from other tissues. Tumortissue blocks were processed for H & E staining, which confirmedthat >95% of the tissue examined comprised tumor cells free of dermal orsubdermal tissue. The levels of PTEN in tumor tissue and numerous normaltissues within the athymic nude mouse were compared. Using anti-PTENantisera, the expression of PTEN was detected in all tissues, with theexception of skeletal and heart muscle (data not shown). No PTEN wasdetected in parental U87-derived tumor tissue (FIG. 2C, lane 4). Theseresults demonstrate that the tumor tissue sampled containedpredominantly tumor cell-derived proteins. As observed in the cell linesgrown in vitro, subcutaneous tumors derived from U87 cells reconstitutedwith mutant or wild type PTEN display similar levels of PTEN expression(FIG. 2C, lanes 1, 2, 3 & 5). Phospho-AKT activity was higher inPTEN-null U87 cells and U87 cells reconstituted with R130M and to alesser extent in U87 cells expressing the G129E mutant (FIG. 2C, lane 1)as compared to the wild type PTEN transduced cells (FIG. 2C, comparelanes 1, 2 & 4 to lanes 3 & 5). The pattern of phosphorylated AKT wassimilar when the different U87 mutant expressing cell lines were assayedin vitro or in vivo (compare FIGS. 1A to 2C). Despite the similar invitro growth rate, there was a dramatic difference in the growth oftumors derived from parental U87 cells compared to cells reconstitutedwith wild type PTEN (FIGS. 2A & B). The average volume of U87-derivedtumors on day 25 after implantation was 848±203 mm3, compared to 91±27mm3 for tumors derived from PTEN-reconstituted cells (n=5, p<0.0001).Reconstitution with catalytically dead mutants of PTEN significantlyreduced the rate of growth in vivo without a demonstrable effect onangiogenesis (FIGS. 2A and 3C). Others have observed effects ofcatalytically dead PTEN expression on cell invasion, suggesting afunction for other regions of the PTEN molecule in cellular functions.Interestingly, in vivo BrdU labeling of tumor cells revealed nosignificant difference in number of cells in S phase (72±6 BrdU positivecells per field in parental U87MG tumor mass versus 68.5±3 in WT PTENreconstituted tumors). These data demonstrated that the loss ofPTEN-mediated inositol phospholipid phosphatase activity was a criticalcomponent of deregulated tumor growth. Notably, the G129E mutant (whichlacks inositol phospholipid phosphatase activity) was equivalent to theR130M mutant (which lacked inositol phospholipid and phosphoproteinactivity) with respect to both tumor growth and the proliferative rateof these tumors in vivo.

[0130] To assess the effect of PTEN on angiogenesis, parental U87 cellswere compared to cells reconstituted with wild type or mutant PTEN.Cryostat sections from subcutaneous tumors for were stained for CD31(PECAM), an endothelial marker used to measure the microvessel densityof these tumors. Microvessel density was assessed from multipledigitized images of CD31-stained tumor tissue at 100× magnification (3fields were evaluated per tumor) and counted blindly for the number ofCD31 positive microvessels per unit surface area as described (Weidneret al 1991 N Engl J Med 324:1-8). Reconstitution of PTEN expression inU87 cells dramatically suppressed the angiogenic response in vivo (FIGS.3A & B). Quantitation of microvessel density in tumors derived fromparental U87 cells (77±13) and U87 cells expressing wild type PTEN(38±7) revealed an 50% suppression of angiogenesis (FIG. 3C) (n=5,p<0.001). The microvessel density of tumors derived from U87 cellsreconstituted with catalytically impaired PTEN (R130M, 84±15 or G129E,69±16) were not significantly different (p>0.05) from the parental U87cell line (FIG. 3C). Similar results were obtained from an analysis ofmicrovessel density of intracranial tumors. The levels of phospho-AKTdetected within the tumor mass in vivo demonstrated a mechanistic linkbetween the loss of the inositol lipid phosphatase function of PTEN, thephosphorylation status AKT and the angiogenic phenotype within thetumor.

[0131] Recent in vitro data suggested a link between PTEN and downstreamtargets including AKT, HIF1α and VEGF in the potential control ofangiogenesis (Zundel et al., Genes Dev 2000, Zhong et al., Cancer Res2000). We used the RNase protection assay (RPA) to examine the effect ofPTEN on thrombospondin 1 (TSP-1) expression. RPA was performed with aTSP-1 specific probe in U87MG cells constitutively expressing wild typePTEN or G129R PTEN (FIG. 3D). The data demonstrate that wild type butnot mutant PTEN expression induces TSP-1 in U87 cells. To confirm theseresults, Western blot analysis was performed to assess TSP-1 expressionin a retroviral-based ecdysone-inducible PTEN expression system (No etal PNAS 1996). Inducible and dose-dependent expression of PTEN wasconfirmed in U87 cells. The induced expression of wild type PTEN, butnot G129R PTEN, resulted in augmentation of thrombospondin 1 expression(FIG. 3E) and suppression of AKT activation as demonstrated by decreasedphospho-AKT levels without an accompanying decrease in total AKT (datanot shown). The induced expression of mutant G129R had no effect onphospho-AKT levels. The data therefore demonstrate that PTEN positivelymodulated the expression of thrombospondin 1, a negative regulator ofangiogenesis (FIG. 3D and E) (Sheibani and Frazier, (1999) Histol.Histopathol. 14:285-294, Hsu et al., (1996) Cancer Res. 56:5684-5691).These data suggested that PTEN has a pivotal role in angiogenesismediated, in part, by the induction of TSP-1.

[0132] Vascular endothelial growth factor (VEGF) is a known positiveregulator of angiogenesis (Jiang et al. (2000) Proc Natl Acad Sci U S A,97(4), 1749-53; Mazure et al. (1997) Blood, 90(9), 3322-31; Mazure etal. (1996) Cancer Res, 56(15), 3436-40; Plate et al. (1994) Int JCancer, 59(4), 520-9; Plate et al. (1992) Nature, 359(6398), 845-8). Thecapacity of tumor tissue to produce VEGF was determined for U87MGparental cells null for PTEN versus U87 cells reconstituted with wildtype PTEN or mutants of PTEN (G129E or R130M). Tumor tissue obtainedfrom subcutaneously implanted tumor cells was subjected to cryostatsectioning and multiple sections through the tumor tissue were pooledfor biochemical Western blot analysis using anti-VEGF antibody (SantaCruz, SC-507). Cell lysates were assayed for protein concentration byBradford method. Equivalent amounts of total protein were resolved bySDS PAGE followed by immunoblot for VEGF protein.

[0133] The results demonstrate that the reconstitution of wild type PTENbut not catalytically dead PTEN markedly suppresses the production ofVEGF by U87MG tumors in vivo. No VEGF was detected in tumorsreconstituted with the wild type PTEN (FIG. 4, lanes 3 and 4). Anintermediate level of suppression is noted in tumors reconstituted withthe G129E mutant of PTEN (FIG. 4, lane 1), a mutant which has lost itscapacity to dephosphorylate PIP₃ and not protein substrates. These datashow the first evidence that PTEN suppresses VEGF, a proangiogenicgrowth factor in vivo. The data implicate PTEN and therefore, the PI-3kinase cascade in the coordinate regulation of the “angiogenic switch”mechanism which controls angiogenesis under normal physiologicconditions. It is this control that is lost during tumor progression.

[0134] Brain tumor-induced angiogenic responses are known to occur inthe context of brain specific stromal and extracellular matrixinteractions. To determine whether the expression of PTEN affected thesurvival of mice in an orthotopic brain tumor model, U87 cellsexpressing either wild type or mutant forms of PTEN were implanted understereotactic control into the right frontal lobe of nude mice (FIGS.5A-D, see arrow for site of implantation). The results demonstrated thatreconstitution of wild type PTEN in U87 cells suppressed the malignantpotential of these cells in an orthotopic animal model. Thus, there was90% survival at 40 days in animals implanted with the wild typePTEN-reconstituted U87 cells compared to 100% mortality of miceimplanted with the parental cells at 27 days (FIG. 5E) (n=15, p<0.0001).PTEN reconstituted tumor cells grew more slowly when implanted in thefrontal lobe (FIG. 5, compare A & B) and remained circumscribed to thatarea of brain (data not shown). U87 cells reconstituted with PTENmutants, ablated for either inositol lipid phosphatase activity (G129E)or all phosphatase activity (R130M), displayed a phenotype similar tothe PTEN-negative, parental U87 cells (FIG. 5C). Animals with tumorsderived from U87 cells reconstituted with PTEN-G129E displayed slightlyprolonged survival (50% at day 30) compared to those implanted withparental U87 cells; all of these animals were dead, however, by day 40.These data implicate the inositol lipid phosphatase activity of PTEN isrequired for controled angiogenesis (FIG. 3) and its loss is correlatedwith the development of a highly malignant glioma (FIG. 5C) followingimplantation of U87 tumor cells in mice.

EXAMPLE II PTEN Reconstitution Reduces Metastatic Potential of BrainTumor Cells Introduced via the Carotid Artery and Negatively RegulatesProangiogenic Factors

[0135] The following methods are provided to facilitate the practice ofExample II.

[0136] As described in Example I, wild type PTEN or mutant PTEN (G129E,R130M) cDNAs were subcloned into the pBabe-puro retroviral expressionvector. Stable clones of U87MG cells were established under puromycinselection (2 ug/ml) (Myers et al. 1998). A panel of antibodies wereobtained which are immunospecific for PTEN (Myers et al. 1998), AKT,phospho-AKT (New England Biolabs, #9270), TIMP-3, MMP-9, and MMP-2(Sigma).

[0137] To evaluate the role PTEN plays in regulating the expression offactors that promote angiogenesis and invasiveness of glial tumors, anin vitro gelatin zymography assay was performed. Gelatin zymographyspecifically detects the presence of members of the matrixmetalloproteinase (MMP) family of proteins, which degrade the ECM,thereby promoting angiogenesis and metastasis. The assay was performedusing either 1) lysates derived from tumor tissue or 2) conditionedmedia in which the tumor cells or tissue were maintained. Proteinsamples for analysis were generated from isolated subcutaneous tumors bydissolving the tumor tissue in a detergent lysis buffer [50 mM Tris-Cl,(pH 8.0), 150 mM NaCl, 0.05% NP-40, 100 mM NaF, 1 mM EDTA, 1 mM EGTA,0.08 mM PMSF, 0.01 mg/ml leupeptin, 0.01 mg/ml aprotinin, 1 μmg/mlpepstatin A]. Protein samples for analysis were also generated fromtumor cell conditioned media concentrated by centrifugation throughMicrocon® YM-10 centrifugal filter devices. The protein concentration ofsamples composed of either tumor cell lysate or concentrated,conditioned media derived from the various tumor cell cultures wasdetermined by standard protein assay (Bio-Rad, Hercules, Calif.).Protein samples were then normalized for equal protein concentrations.

[0138] Protein samples (10 μg) were subjected to substrate gelelectrophoresis with modifications. Briefly, protein samples normalizedfor protein concentration were applied, under non-reducing conditions,to 10% polyacrylamide slab gels impregnated with 1 mg/ml gelatin(DIFCO). After electrophoresis, the gel was washed at room temperaturefor 30 minutes in washing buffer [50 mM Tris-Cl (pH 7.5),5 mM CaCl₂, 1mM ZnCl₂, 2.5% Triton X-100] and then incubated overnight at 37° C.,with gentle agitation, in washing buffer containing 1% Triton X-100. Thegels were stained with a solution of 0.1% Coomassie brilliant BlueR-250. Clear zones in the gel are indicative of the presence ofgelatinolytic activity contained in a given protein sample. Thegelatinolytic activity was quantitated by densitometric scanning andanalysis.

[0139] To further evaluate the role PTEN plays in regulating theexpression of factors that promote angiogenesis and invasiveness ofglial tumors, Matrigel® invasion assays were also performed. Matrigel®invasion assays are a standard procedure used to characterize themetastatic potential of cells, based on the ability of such cells todegrade the Matrigel® extracellular matrix (ECM). Matrigel® is acommercially available mix of basement membrane components, generatedfrom an EFS sarcoma, which includes basic components of the basementmembrane such as collagens, laminin, and proteoglycans, as well asmatrix degrading enzymes, their inhibitors, and growth factors. Invasionof tumor cells into Matrigel® has been used to characterize involvementof matrix-degrading enzymes which play important roles in tumorprogression and metastasis (Benelli and Albini, 1999).

[0140] The assay was performed as follows: Matrigel® (Becton-Dickinson)was thawed overnight at 4° C. on ice and diluted to 1 mg/ml inserum-free DMEM. 50 μl of the diluted Matrigel® was added to the upperchambers of a 24-well Transwell® plate(0.8 μm pore size, Costar). Theupper chambers were then incubated at 37° C. for 6 hours to facilitatesolidification of the gelatinous matrix. U87 glioma cells were harvestedfrom tissue culture flasks by 0.04% Trypsin/EDTA and washed three timeswith serum-free DMEM to remove trace amounts of sera. Cells wereresuspended in serum-free DMEM at a density of 5×10⁵ cells/ml, 200 μl ofwhich was added to upper chambers coated with solidified Matrigel®. Thelower chambers of the Transwell® plate were filled with 600 μl of DMEMcontaining 5 μg/ml fibronectin, which served as a soluble attractant andadhesive substrate. Transwell® plates were incubated at 37° C. for 36hours to facilitate migration of U87 cells from the upper chambers intowhich they were seeded to the lower chambers. Following this incubationtime, the upper chambers were removed and stained with 0.1% crystalviolet solution to visualize the cells. Cells that failed to migrate tothe underside of the porous membrane that separates the top and bottomchambers of the Transwell® were removed by scraping the topside of themembrane with a cotton-tipped swab. Cells that had successfully migratedthrough the Matrigel® to the underside of the porous membrane werecharacterized as invasive cells and counted under 100× magnification.

[0141] An in vivo study of experimental metastasis following orthotopicintroduction U87 glioma cells into the nude mouse brain was alsoundertaken. U87MG cells were introduced into the circulatory system of amouse via the injection of cells into the intracarotid artery. Eachmouse was anesthetized by intraperitoneal injection of Nembutal andrestrained on a cork board equipped with fixed rubber bands that wereused to wrap around the teeth of the upper jaw, thereby immobilizing thehead. Under a dissecting microscope, the hair over the trachea (if amouse species having hair was used) was shaved, the neck was preparedfor surgery with betadine, and the skin cut by a mediolateral incision.After blunt dissection, the trachea was exposed and the musclesseparated to expose the right common carotid artery, which was thenseparated from the vagal nerve. Further dissection was then performed toreveal the internal and external carotid arteries. The common carotidartery was prepared for injection distal to the point of divisionbetween the internal and external carotid arteries. Briefly, a ligatureof 5-0 silk suture was placed in the distal portion of the commoncarotid artery and a second ligature was positioned and tied looselyproximal to the injection site of the internal carotid artery. A sterilecotton tip applicator was inserted under the artery just distal to theinjection site to elevate the artery. This procedure controlled bleedingfrom the carotid artery by regurgitation from distal vessels. The arterywas nicked with a pair of microscissors, and a plastic cannula (<30gauge) was inserted into the lumen and threaded forward into theinternal carotid artery. Wild type cells or cells expressing the variousmutants of PTEN, G129R, G129E, R130M, (1×10⁶ in 10 μl) resuspended inPBS were injected slowly into the artery, after which the cannula wasremoved. The second ligature was then tightened and the incision in theskin sealed with surgical clips.

[0142] Following completion of the surgery, mice were placed in cleancages equipped with heating sources to maintain their body temperature.The mice were then monitored until they recovered from the effects ofthe anesthesia and returned to the care of the animal facility.Deleterious systemic effects associated with brain lesions, such ascachexia, listlessness, and protrusion of the right bulb were monitored.Mice were sacrificed when moribund. Brains were removed and fixed in 10%formaldehyde for H&E staining and in OCT for frozen sections.

[0143] Cell lysates obtained from U87 cells grown in tissue culture orfrom multiple cryostat sections of U87MG subcutaneous tumor tissues werealso run on gels and analyzed by Western blotting. A Bradford assay wasperformed to determine protein concentration of each lysate. Equivalentamounts of protein were resolved by SDS PAGE and transferred tonitrocellulose. Membranes were probed with antisera specific for PTEN,AKT, phospho-AKT or TIMP-3. The RNAase protection assay (RPA) wasperformed using a RPA III kit from (Ambion) according to the manufacturespecifications. Briefly, 20 μg of total RNA was precipitated andresuspended in 10 μl of hybridization buffer containing specificradioactive probe. The RNA was then heated to 95° C. for 10 min andhybridized for 16 hours at 42° C. 150 μl of this mixture was treatedwith 1:100 dilution of RNAase in RNAase buffer for 30 minutes. TheRNAase was inactivated and the RNA was reprecipitated and resolved on 5%acrylamide gel. RNA probes were synthesized using MAXI Script utilizingPCR templates and T7 polymerase. The GAPDH probe was provided in the kitas an internal control. The TIMP-3 probe represented a 590 nucleotidesequence located in the 3′ UTR of the TIMP-3 sequence. All probes weresequenced.

[0144] Microvessel density (MVD) was determined for each brain tumor asdescribed in Example I by CD31 staining, performed on cryostat sections(7 μm), fixed in acetone, blocked in 1% goat serum and stained withanti-CD31 antibody (Pharmingen, #01951D). Antibody staining wasvisualized with peroxidase-conjugated anti-mouse antibody and counterstained with hematoxylin. A negative control was performed on each tumortissue stained with mouse IgG. Two sections from each tumor were scannedunder low power magnification (40×) to identify areas of highest CD31positive vessel density (Weidner et al. 1991), followed by digitizationof 5 fields from this area. The digitized images representing one 100×field were counted for the number of CD31 positive vascular elements.Data was collected independently by two researchers in a blind study.The average number of microvessels per digitized 100× field wasdetermined for 5 tumors per experimental group and analyzed by Student'st-test.

[0145] The data obtained thus far indicated that the anti-angiogenicactivity of PTEN can be correlated matrix remodeling and degradation. Weused the RNAase protection assay (RPA) to confirm the effect of PTEN onTIMP-3 expression. RPA was performed with a TIMP-3 specific probe inU87MG cells constitutively expressing wild type PTEN or G129R PTEN. Thedata demonstrated that wild type, but not mutant PTEN expression inducedTIMP-3 expression in U87 cells. Thus, PTEN positively modulates theexpression of TIMP-3, a negative regulator of matrix metalloproteinases(FIG. 6). These data reveal a novel mechanism through which PTENregulates angiogenesis and extracellular matrix remodeling via theinduction of TIMP-3 and suppression of matrix metalloproteinase levels.To confirm this observation, matrix metalloproteinase activity in PTENnull and wild type PTEN reconstituted tumors (FIG. 7), was examined byperforming reverse zymography for collagenolytic activity. Consistentwith the finding that PTEN induced TIMP-3 expression in U87 tumor cells,it was apparent that wild type PTEN reconstitution suppressed MMP-9activity with no effect upon MMP-2 activity (FIG. 7) (Oh et al. 1999;Rao et al. 1994).

[0146] To determine the role PTEN play is tumor cell invasion, PTENdeficient and PTEN reconstituted glioma cells were evaluated for theircapacity to invade and migrate through a matrigel coated membrane. PTENreconstitution was observed to completely abrogate invasion of U87 tumorcells through matrigel (FIG. 8). Adhesion and migration on matrigelremained intact in PTEN reconstituted U87MG cells (data not shown).Importantly, the data provided the first direct evidence that PTENcontrols matrix degradation and the invasive behavior of tumor cells invivo, and suggest that PTEN as well as PI-3 kinase inhibitors willsuppress tumor invasion and the metastatic phenotype in vivo.

[0147] The data described above clearly demonstrate that mutations inthe PTEN tumor suppressor in U87 glioblastoma cells resulted in the lossof normal physiologic control of matrix remodeling and invasion.Furthermore, analysis of the data revealed that PTEN exerts its effectat the level of TIMP-3 expression and control of MMP-9 matrixmetalloproteinase activity. The results, therefore, implicate PI-3kinase and other downstream targets of PTEN such as AKT, in the controlof metastasis and invasion of tumor cells. The U87 brain tumor modeltherefore provides an ideal assay system to identify agents thatnegatively and/or positively regulate the activities of PTEN or othersignaling molecules in the pathway such as VEGF, or bFGF, etc. whichcontrol matrix degradation in vivo. Finally, the results provided thefirst direct evidence that PTEN controls the capacity of a tumor todegrade the extracellular matrix in vivo. As mentioned previously, PTENpossesses lipid phosphatase activity which preferentiallydephosphorylates phosphoinositides at the D3 position of the inositolring. To date, PTEN is only one of two enzymes reported to have thisactivity. Reconstitution of PTEN in tumor cells that carry mutations inthe PTEN gene, have established that this phosphatase regulates the PI-3kinase-dependent activation of AKT, a major player in cell survival.This observation indicates that PTEN may function as a direct antagonistof PI-3 kinase and PIP-3 dependent signaling. Accordingly, PI-3 kinaseinhibitors, such as LY294002, should be efficacious in blocking matrixdegradation, invasion and metastasis of tumors in vivo. Finally, sincethese pathways are also important in wound healing, the data provide thefirst mechanistic link between PI-3 kinase signaling pathways and PTENsignaling pathways in the control of wound healing and wound associatedangiogenesis.

EXAMPLE III Inflammatory Signaling Downstream of Fc Receptor Activationis Modulated by Agents that Alter Tyrosine Kinase and PhosphataseActivity

[0148] Fc gamma receptor mediated phagocytosis is a model forimmunoreceptor (ITAM) signaling and involves the activation of proteintyrosine kinases and protein tyrosine phosphatases. Relatively little isknown of role of lipid phosphatases in control of ITAM signaling andinflammation. We used phagocytic J774A.1 cells and a heterologous COS7cell system to examine the roles played by Src family of proteintyrosine kinases, Syk and PI-3 kinase and protein tyrosine phosphatase,PTEN in signal transduction pathway leading to Fcγ receptor mediatedphagocytosis. Heterologous expression of dominant negative Syk inJ774A.1 cells significantly inhibited phagocytosis of sensitized sRBCsand preincubation of the cells with the Src specific protein tyrosinekinase inhibitor, PP1, and PI-3 kinase inhibitor, Wortmannin, was shownto inhibit this response. Stimulation of J774A.1 cells with sensitizedsRBCs induced tyrosine phosphorylation of Cbl, which was inhibitedsignificantly by PP1 and to some extent by heterologous expression ofdominant negative Syk implicating Src and syk in the phosphorylation ofCbl in phagocytic signal transduction. The heterologous overexpressionof PTEN completely abrogated the phagocytosis of IgG sensitized sheepred blood cells (sRBCs) compared to catalytically inactive mutant ofPTEN. These data provide the first evidence that PTEN, a tyrosinephosphatase, is involved in the regulation of Fcγ receptor mediatedphagocytosis and the regulation of immunoreceptor tyrosine basedactivation motif (ITAM) based signaling events in hematopoietic cells.

[0149] Overexpression of a catalytically dead PTEN phosphatase resultedin augmented phosphorylation of AKT and augmentation of phagocytosis andITAM signaling. These results show that activating signals provided bySrc family kinases, Syk and PI-3 kinase are opposed by inhibitorysignals through PTEN in the control of Fcγ receptor mediatedphagocytosis. Thus, PTEN agonists and PI-3 kinase inhibitors shouldprovide potent anti-inflammatory and immunosuppressive agents to controldownstream signaling events mediated by ITAM linked receptors found inhematopoeitic cells (e.g., T cell receptor, B cell receptor, Fcreceptors and collagen receptor VI). Such agents should also exertpotent control over the immune responses mediated through T cells, Bcells, myeloid cells including macrophages, neutrophils, dendriticcells, monocytes, mast cells and platelets by preventing theiractivation and subsequent modulation of unwanted immune reactivity andplatelet aggregation which contribute to a number of human diseases.

[0150] The following methods and materials are provided to facilitatethe practice of Example III.

[0151] Anti-Cbl antibody was obtained from Santa Cruz Biotechnology,Santa Cruz, Calif. Anti-Syk antibody was provided by Dr. Tamara Hurley(The Salk Institute, San Diego, Calif.) and anti-PTEN antibody wasgenerated by immunizing rabbits with an N-terminal peptide of PTEN.

[0152] J774A.1, a macrophage-like cell line, and Cos 7 cells wereobtained from the ATCC. Both cell lines were maintained in DMEMsupplemented with 10% fetal calf serum (FCS). Recombinant vaccinia virusvectors were provided by Dr. Bernard Moss (National Institutes ofHealth, Bethesda, Md.). The dominant negative Syk vaccinia construct(encoding the amino terminal residues 1-255 of Syk) which also encodesbeta-galactosidase was provided by Dr A. Scharenberg (Sharenberg et al.,(1995) Embo J. 14:3385). Recombinant vaccinia viruses containing PTENand dominant negative Syk were prepared as described below.

[0153] Briefly, recombinant vaccinia viruses were propagated in 149Bcells grown in RPMI medium containing 10% FCS. A confluent culture ofcells was infected with recombinant vaccinia virus at a concentration of0.5 pfu/cell for 48 h. The cells were scraped from the plastic in thesame medium, centrifuged to generate a cell pellet, and resuspended in 5ml of 10 mM Tris-HCl (pH 9). The cells were lysed by three cycles offreezing in liquid nitrogen and thawing at 37° C., after which thevolume of the cell lysate was adjusted to 20 ml with 10 mM Tris-HCl (pH9) in preparation for mechanical lysis step provided by forty strokes ina homogenizer. Nuclei and cell debris were separated from the celllysate by centrifugation at 1,000 rpm for 5 min. The cell lysatecontaining the recombinant vaccinia virus was then subjected tosonication for 1 min with 50% output and a 30% duty cycle. The celllysate was loaded on a cushion of 36% sucrose solution and centrifugedat 13,000 rpm for 80 min 4° C. in an ultracentrifuge (Beckman, PaloAlto, Calif.) using a SW.28 rotor. Viral pellets obtained wereresuspended in 1 ml of 10 mM Tris-HCl (pH 9) and loaded onto a sucrosegradient composed of 6.6 ml each of 40%, 36%, 32%, 28% and 24% ofsucrose solutions made in 10 mM Tris-HCl (pH 9) to be centrifuged at12,500 rpm for 50 min at 4° C. in an ultracentrifuge using a SW.28rotor. A bluish white ring containing purified virus was collected anddiluted with 10 mM Tris-HCl (pH 9) prior to a final centrifugation at13,000 rpm for 60 min at 4° C. in an ultracentrifuge using a SW.28 rotorto pellet the virus. Purified recombinant vaccinia virus thus obtainedwas suspended in 10 mM Tris-HCl (pH 9) and titered as followed. Analiquot was used for generating serial dilutions of the concentratedviral suspension. The serial dilutions were used to infect a confluentlawn of 149B cells grown in 35 mm wells for 2 h at 37° C. in 1 ml ofRPMI medium containing 10% FCS. The medium was then replaced with 3 mlof fresh RPMI medium containing 10% FCS. The medium was discarded after24 hours incubation and viral plaques were visualized by staining withcrystal violet to titer the virus.

[0154] A DNA construct encoding the catalytically dead trap mutant ofPTEN, C124S, was kindly provided by Nicholas Tonks (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). This mutant The PTEN insert wasamplified by PCR using the following 5′ and 3′ primers:5′GG-GTC-CAC-ATG-ACA-GCC-ATC-ATC-AAA-GAG′3′ forward primer (SEQ ID NO:19) and 5′-GG-TCT-AGA-TCA-GAC-TTT-TGT-AAT-TTG-TGA′3′ reverse primer (SEQID NO: 20),

[0155] respectively. The amplified product was subcloned into the PCR2.1vector for propagation using the TA cloning kit (Invitrogen, San Diego,Calif.), and the PTEN insert was cleaved from the vector by digestionwith SmaI and SalI and subsequently ligated into a linearizedrecombinant vaccinia vector pSC65 to generate pSC65-PTEN. The constructC2pSC65 was used to make a recombinant vaccinia virus using thepackaging cell line CV1 and the wild type vaccinia virus. Recombinantvirus was isolated from wild type virus by single plaque purification,and then amplified, purified, and titered as described above.

[0156] J774A.1 phagocytic cells were plated at 2×10⁵ cells per well in atwelve well plate (Costar, Corning, NY) and cultured overnight. Cellswere infected with recombinant vaccinia virus pSC65 or pSC65-PTEN at adensity of 2 pfu/cell for 4 h at 37° C. in 5% CO₂. The media was changedafter 4 hours and the cells were incubated with sheep red blood cellscoated with IgG at a subagglutinating concentration. The target toeffector ratio was 100:1. The cells were harvested after 2 hours, andcytospins were prepared, fixed, and stained with Wright Giemsa stain(Dade, AG, Switzerland). Stained slides were evaluated microscopicallyfor rosette formation. The remaining uningested sRBCs were subjected tolysis by osmotic shock in water. The cells were suspended in DMEM mediumcontaining 20% FCS. The cells were spun down on glass slide and fixedand stained by Wright Giemsa stain. A minimum of one hundred and fiftycells were counted for each slide and the phagocytic index wascalculated as follows: Phagocytic Index (PI)=% of phagocytic cells xaverage number of sRBCs engulfed by each cell.

[0157] In the drug inhibition studies, the cells were subjected totreatment with an indicated inhibitor at different concentrations alongwith an appropriate DMSO control for 1 hour in DMEM with 10% FCS beforecarrying out the phagocytic assay.

[0158] Assays to detect β-galactosidase activity were performed asfollows: cells (1×10⁵) were suspended in 400 μl of DMEM containing 10%FCS to which 50 μl of 1% X-gal (Sigma, St.Louis, Mo.) was added.Subsequent incubation at 37° C. facilitates β-galactosidase activitywhich is indicated by acquisition of a blue color by the reactionmixture. The reaction mixture was diluted 1:10 and the optical densitywas measured at 595 nm in a spectrophotometer (Molecular Devices, MenloPark, Calif.).

[0159] J774A.1 cells and COS7 cells (2×10⁵) infected with recombinantviruses expressing either dominant negative Syk or PTEN were lysed in 50μl of sample buffer. The lysates were resolved by SDS-PAGE, transferredto a solid matrix support, and probed to assess protein expression withspecific antibodies.

[0160] J774A.1 cells were infected with recombinant vaccinia virus atthe concentration of 2 pfu/ml for 4 hours. The infected cells were thenpelleted by centrifugation, and resuspended at a concentration of 2×10⁶cells per ml in DMEM to be stimulated with IgG coated sRBCs at 37° C.for 5 min. The samples were pelleted at 500× g in a refrigeratedcentrifuge and the resultant cell pellet was lysed as described earlierand analyzed following immunoprecipitation with specific antibodies.

[0161] Dominant Negative Syk Inhibits Phagocytosis

[0162] In order to investigate the role played by the nonreceptortyrosine kinase Syk in IgG mediated phagocytosis, a dominant negativemutant form of Syk was expressed in J774A.1 cells utilizing recombinantvaccinia virus as a means of transmission. This Syk mutant encodes atruncated form of Syk which comprises the tandem SH2 domains, butexcludes the catalytic domain. As a result of this mutation, the proteinbehaves as a dominant negative mutant by binding to the ITAMs of theFcyR subunit, thereby blocking the interaction of the endogenouscatalytically active Syk with these sites. The results demonstrated thatthe expression of dominant negative Syk in J774A.1 cells inhibitedphagocytosis of IgG coated sRBCs (FIG. 9A). In contrast, J774A.1 cellsinfected with empty vector recombinant vaccinia virus, as a control,engulfed sensitized sRBCs normally. The expression levels of dominantnegative Syk in infected cells was evaluated by Western analysis usingantibodies specific for Syk (FIG. 9B, lane 3). To ensure that theinhibitory effect on phagocytosis was specific to the expression ofdominant negative Syk, and not a consequence of differential levels ofviral infection, infected J774A.1 cells were assessed for viral load.Since the pSC65 plasmid, into which the dominant negative Syk wascloned, also contained the gene encoding β-galactosidase, this enzymewas used as an internal control for viral expression levels. Briefly,β-galactosidase activity can be quantified calorimetrically as indicatedby the appearance of a colored product following cleavage of thesubstrate X-gal. In every experiment, the levels of recombinant viralload were equivalent to those of control cells (infected with emptyvector recombinant vaccinia virus) and experimental cells (infected withvaccinia virus containing dominant negative Syk; data not shown).

[0163] As an additional control to evaluate the specificity of theinhibition observed after expression of dominant negative Syk, thecapacity of J774A.1 cells to form rosettes via the FcyR was examinedfollowing infection with different viruses. Cells infected with emptyvector recombinant vaccinia virus and cells infected with vaccinia viruscontaining dominant negative Syk displayed 100% rosette formation within1 minute of addition of sensitized sRBCs; these data indicated thatneither the cell surface expression level of FcγRs nor their bindingcapacity for sRBC targets was affected by infection with recombinantvaccinia virus alone (data not shown). Notably, rosette formation andphagocytosis did not occur in the absence of sensitizing antibodyagainst sRBCs, thereby further underscoring the specificity of theseresponses. These data using dominant negative Syk were consistent withother data in the literature, including those derived from Syk knockoutmice (Crowley, M., et al., (1997) J. Exp. Med. 186:1027), which stronglysupport a role for Syk in propagating signals required for IgG mediatedphagocytosis in the J774 system.

[0164] Src and PI-3 Kinase are Required for Phagocytosis of IgG CoatedsRBCs by J774A.1 Cells

[0165] Recent evidence from Hck,/Lyn/Fgr knockout mice suggests thatmembers of the Src family of nonreceptor protein tyrosine kinasesfunction upstream of Syk and PI-3 kinase in ITAM signaling (Crowley etal., 1997, supra). To examine the role of Src in FcyR-mediatedphagocytosis, J774 cells were treated with different concentrations ofPP1 (1, 5, or 10 μM; Calbiochem, La Jolla, Calif.), a Src familytyrosine kinase inhibitor, wortmannin(1 or 5 μg/ml), an inhibitor ofPI-3 kinase (LY294002), or DMSO as a control. Briefly cells were treatedwith the above reagents for 1 hour in DMEM supplemented with 10% FCS andthen sensitized sRBCs were added at a target to effector ratio of 100:1.As shown by FIG. 10A, PP1 inhibited phagocytosis in a dose dependentmanner and completely abrogated phagocytosis at a 10 μM concentration.As shown in FIG. 10B, 5 μg/ml wortmannin also mediated significantinhibition of phagocytosis. These observations implicate the Src kinasefamily and PI-3 kinase in IgG-mediated phagocytosis of sRBCs by J774A.1cells.

[0166] Effect of Dominant Negative Syk and Src Inhibitor, PP1, onTyrosine Phosphorylation of Cbl in Response to Stimulation withSensitized sRBCs

[0167] It is well known that Fcγ receptor crosslinking induces thetyrosine phosphorylation of the adapter protein, Cbl (Park, R. K., etal., (1996) J. Immumology 160:5018). To determine if phagocyticsignaling events lead to the phosphorylation of Cbl, the degree of Cblphosphorylation was assessed before and after induction of phagocytosis.To investigate the role of specific kinases in this phosphorylationevent, dominant negative Syk and the Src family kinase inhibitor PP1were utilized to inhibit the activity of these enzymes. The resultsdemonstrated that Cbl was phosphorylated on tyrosine residues followinginduction of phagocytosis and this phosphorylation event was abrogatedby PP1 (FIGS. 11A and 11B, compare lanes 2-3 to 5-6). This effect wasdose dependent (data not shown), as was the effect of PP1 on inhibitionof Fcγ receptor-mediated phagocytosis (FIG. 11A). Interestingly,dominant negative Syk inhibited Cbl tyrosine phosphorylation to a lesserextent but completely abrogated the phagocytic response. Interestingly,both PP1 and dominant negative Syk suppressed the basal tyrosinephosphorylation levels of Cbl in vivo. These data suggested that thecatalytic activity of the Src family kinases and the capacity of Syk todock with the ITAM receptor were both required for Cbl phosphorylationin response to phagocytic stimuli and that these two events wererequired for phagocytosis. The dominant negative Syk would not beexpected to alter the upstream activity of Src family kinases and henceSrc mediated phosphorylation of Cbl was not altered to the same extent.The data provided support for a signaling cascade in which Syk functionsdownstream of Src and upstream of Cbl and other effectors associatedwith Cbl such as the p85 subunit of PI-3 kinase. The data demonstratedthat Src family kinases mediated the phosphorylation of Cbl in a Sykkinase independent manner in vivo. The data also revealed that Srcfamily kinases and Syk were required for phagocytosis mediated by thedownstream activation of PI-3 kinase.

[0168] Of note, more recent data identified the tyrosine residue atposition 731 of Cbl as a consensus binding site (YxxM; SEQ ID NO: 21)for the p85 regulatory subunit of PI-3 kinase. Upon phosphorylation,this motif was recognized as a target for PTEN (data not shown). Hence,PTEN plays a signaling role in regulation of ITAMs action on PI-3 kinaseto control ITAM signaling events.

[0169] Overexpression of PTEN in COS7 System Inhibits FcγRIIA ITAMSignaling

[0170] Since tyrosine kinases are required for phagocytosis and lead tothe activation of PI-3 kinase which phosphorylates phospholipids thatact as second messengers, dephosphorylation of such phosphoinositidesmay serve a role to downregulate this response. To address this issue,J774A.1 and COS7 cells were genetically engineered to overexpress thedual specificity phosphatase PTEN, which is known to dephosphorylatePIP3, a critical phosphoinositide second messenger. Overexpression ofPTEN in COS7 cells markedly inhibited phagocytosis of IgG coated sRBCs(FIG. 12). PTEN expression reduced the phagocytic index (FIG. 12) by 95%as compared to that of control cells. In contrast, the C124S mutant ofPTEN, which is catalytically dead and can act as a substrate trap,augmented the phagocytic index by a factor of 2.5. See FIG. 12. Thegreen bars represent the percent of the total cell population which wasphagocytic for at least one sRBC. These results demonstrated that PTENnegatively regulated IgG-mediated phagocytosis and that the catalyticactivity of PTEN was required for this suppression.

[0171] These data provide further evidence for the role of PTEN in theregulation of ITAM-mediated signaling in a variety of signaltransduction cascades essential for the propagation of the inflammatoryresponse in T cells, B cells and myeloid cells. Accordingly, suchbiological processes provide the basis for methods for screening andidentifying therapeutic agents which regulate these inflammatoryresponses.

[0172] Our observations suggest that the protein tyrosine kinases, Hckand Syk and PI-3 kinase are activated during FcyR mediated phagocytosis.Stimulation of macrophage cells with IgG coated particles activatesFcγRs, FcγRI and FcγRIII in turn activate Hck/Src kinase whichphosphorylates tyrosine in the ITAM motif in gamma chain associated withthese receptors. This provides a site for attachment and activation forSyk, which would activate the pathway downstream leading to subsequentactivatation of PI3-kinase which is needed for proper cytoskeletalassembly. On the other hand aggregation of FcγRIIB stimulatesphosphorylation of tyrosine in its ITIM motif to provide a site ofattachment and activation for phosphatases like PTEN and SHIP which inturn regulate the pathway downstream.

[0173] Consistent with our data using wortmannin, the 5′ inositidephosphatase, SHIP, serves as a negative regulator of AKT phosphorylationand controls FcyR phagocytosis. Maeda et.al have provided evidence forthe existence of two opposite signaling pathways upon aggregation ofpaired immunoglobulin receptors PIR-A and PIR-B (Maeda et al. (1998) J.of Exp. Med. 188:991) PIR-A induces the stimulatory signal by using ITAMin the associated γ chain while PIR-B mediates the inhibitory signalthrough its ITIM. Our results are consistent with the model that duringFcγ receptor mediated phagocytosis in J774A.1 mouse macrophages theFcγRI and FcγRIII utilize the ITAM motif to send a positive signalinside the cell for phagocytic pathway; whereas FcγRIIB controls thepathway by starting a negative regulatory loop via the ITIM motifthrough PTEN. Accordingly, Syk and Src inhibitors should suppress ITAMimmunoreceptor signaling and control inflammation in concert with PI-3kinase antagonists. It is also likely that synergy will exist whenagents which inhibit more than one ITAM pathway are combined in humans.

[0174] In summary, the data support the involvement of Syk, Src familykinases and phosphatidyl inositol 3-kinase in positive regulation of Fcγreceptors mediated phagocytosis in our system. Described herein is thefirst evidence for the involvement of the protein/lipid phosphatase,PTEN in the negative regulation of phagocytosis and ITAM signaling.These data provide the basis for screening and identification oftherapeutic agents that modulate PTEN, PI-3 kinase or the signalingpathways downstream of PI-3 kinase (PDK-1, PAK, AKT, forkhead, etc).Inhibitors of ITAM propagated signals (e.g., 1) T cell receptor/CD3complex signaling in T cells; 2) B cell receptor signaling in B cells;3) ITAM receptor signaling including Fcγ receptors which mediate myeloidinflammatory diseases through the production of inflammatory cytokines,TNFa, IL-1, IL-4 etc.; 4) the FCεRI receptor in mast cells responsiblefor atopic disease and allergy; and 5) the FcαRI involved in mucosalallergic responses) should effectively regulate the immune responsepathway. Hence PTEN agonists and PI-3 kinase inhibitors shouldeffectively modulate immunopathologic states in animals. Likewise Sykand Src kinase inhibitors should effect ITAM myeloid signaling.

EXAMPLE IV PTEN and PI-3 Kinase Signaling Cascade Regulates p53 andTumor-Induced Angiogenesis

[0175] As described in Examples I, II and III, PTEN regulates thetumor-induced angiogenic response and thrombospondin expression in amalignant glioma model. A recent report by Sabbatini et al suggests aconnection between the PI-3 kinase cascade and the regulation of p53signaling (Sabbatini, 1999; J. Biol. Chem. 274:24263). Activation ofPI-3 kinase/AKT pathways results in the suppression of p53 dependentapoptotic pathways giving rise to conditions that are permissive forcell division. These data suggest a molecular mechanism for thecoordination of signals coming from growth factor receptors through PI-3kinase cascades which would jointly regulate apoptosis, proliferationand recruitment of a new blood supply (neovascularization/angiogenesis).This signaling pathway appears to be tightly regulated in normaltissues. During malignant transformation, this coordinated regulation ofcellular signaling is lost. In the present example, we demonstrate thatPTEN plays a role in coordinating these signaling events within thecell. We also show that loss of PTEN leads to deregulation and tumorprogression. Bearing in mind the link between PTEN phosphatase activityand PI-3 kinase signaling pathways, we performed assays to determinewhether PI-3 kinase inhibitors were capable of reestablishing thisregulatory feedback system thereby restoring normal coordination of cellgrowth and angiogenesis.

[0176] Thrombospondin 1 (TSP-1), angiostatin, endostatin, tissueinhibitors of metalloproteinases (TIMPs) are potent inhibitors ofangiogenesis (Dameron, et al.,(1994) Science 265:1582; Good et al.,(1990)PNAS 87:6624). Malignant brain tumors are known to undergo a morerobust angiogenic response as compared to their benign low-gradecounterparts, and are classified histopathologically by the presence orabsence of high microvessel counts (microvessel density)(MVD).Regulation of PI-3 kinase-dependent signals, including activation of AKTby Vascular Endothelial cell Growth Factor and its receptors, theprotein tyrosine kinases Flt-1 and KDR, have been implicated in braintumor angiogenesis (Plate et al.,(1992) Nature 359:845). Jiang et aldemonstrated in the chicken chorioallantoic membrane model that PI-3kinase-dependent pathways may regulate angiogenesis and VEGF expressionin endothelial cells (Jiang et al., (2000) PNAS 97:1749).Immunohistochemical studies in prostate tumor specimens havedemonstrated that tumors containing PTEN mutations have highermicrovessel counts than tumors expressing wild type PTEN (Giri et al.,(1999) Hum. Pathol. 30:419).

[0177] To test the hypothesis that PTEN is connected to p53transcription we performed experiments in U87MG glioma cell lines whichare wild type for p53 and deficient in PTEN. We conditionally expressedin these U87MG cells, wild type PTEN or catalytically defective mutantsof PTEN to determine if PTEN regulates p53 transcription. We thenperformed experiments with the PI-3 kinase inhibitor using the parentalU87MG cells to determine if LY294002 control over PIP₃ metabolism wouldprevent tumor growth and block angiogenesis in vivo.

[0178] The following protocols are provided to facilitate the practiceof Example IV.

[0179] The constructs and encoding the PTEN mutants have been previouslydescribed in Example I. Tumor implantation methods are also provided inthe previous examples.

[0180] Treatment of Mice with LY294002.

[0181] LY294002 was administered at a dosage of 100 mg/kg delivereddaily by intraperitoneal injection in a small volume of 100% DMSO for 2weeks, beginning 2 days after tumor implantation. No untoward effectswere noted in mice treated with either LY294002 or DMSO. Control micewere injected with small volume (10 ml) of 100% DMSO. Daily measurementof tumor volume was performed in 3 coordinates using calipers.

[0182] Biochemical Analysis.

[0183] Immunoblots were performed on cell lysates obtained from U87cells grown in tissue culture or from multiple cryostat sections ofsubcutaneous tumor tissues. A Bradford assay was performed to determineprotein concentration of each lysate. Equivalent amounts of protein wereresolved by SDS PAGE and transferred to nitrocellulose. Membranes wereprobed with antisera specific for PTEN, AKT, phospho-S473-AKT. We used awell characterized mdm2 promoter linked to firefly luciferase (mdm2lucinserted into the pGL2 vector) that contains p53 DNA binding elements(Ouchi et al., (1998) PNAS 95:2302) to study p53 specific transcriptionin U87 cells under muristirone induced PTEN expression conditions.Another construct, pGL2, contained the mdm2luc promoter which is deletedfor p53 response element was used as a negative control. Cells werecotransfected with pRSVβgal to normalize mdm2 luciferase activity fortransfection efficiency. The Tropix-galacto-light kit and Promegaluciferase assay system was used to quantitate β-galactosidase andluciferase activity, respectively.

[0184] Immunohistochemical and Histopathology.

[0185] Microvessel density (MVD) was determined for as described in theprevious examples.

[0186] Results

[0187] The previous examples demonstrated that the muristirone inducibleexpression of PTEN in U87 cells results in increased levels ofthrombospondin 1 expression, a negative regulator of angiogenesis. Inthe present example, we demonstrate that wild type PTEN suppressed theactivation of phospho-AKT without affecting total AKT (FIG. 13). Theinduced expression of mutant G129R had no effect on phosphoAKT (FIG.13). The data presented in the previous examples indicate that PTEN mayregulate angiogenesis through the induction of TSP-1. One transcriptionfactor that upregulates TSP-1 is the tumor suppressor protein p53(Dameron, (1994) Science 265:1582). We sought to determine if there wasa link between PTEN, TSP1 and p53.

[0188] The data presented herein demonstrate that PTEN regulates p53transcription (FIG. 14). The induction of wild type PTEN and not mutantPTEN induced p53 dependent transcription in U87 cells (7.5-foldinduction)(FIG. 14). Muristirone induced expression of wild type PTEN orG129R protein was equivalent whereas G129E induction was slightlygreater (see insert, FIG. 14). Controls were performed using anmdm2luciferase construct deleted in critical p53 binding sites toconfirm the specificity for PTEN induction of p53 specifictranscription. These data provide compelling evidence that PTEN and p53are linked in a common pathway and therefore provide new biochemicaltargets for influencing PTEN regulation of expression of TSP-1 throughthe regulation of p53 transcription.

[0189] To determine whether PI-3 kinase exerts control over angiogenesisor the growth of glial tumors in vivo the orthotopic brain tumor modeldescribed in Example I was utilized. To assess the effect of LY294002 onangiogenesis, we treated mice with LY294002 (100 mg/kg/dose×2 weeks) orDMSO as negative control. Tumor volumes were recorded daily (FIG. 15).On day 14 we stained cryostat sections from subcutaneous tumors for CD31(PECAM). CD31 is an endothelial marker used to measure the microvesseldensity of these tumors. Microvessel density was assessed from multipledigitized images of CD31-stained tumor tissue at 100× magnification (3fields were evaluated per tumor) and counted blindly for the number ofCD31 positive microvessels per unit surface area as described (Weidneret al., (1991) N. Engl. J. Med. 324:1). Quantitation of microvesseldensity in tumors treated with DMSO versus LY294002 are shown in FIG.16. Compared to controls (FIG. 16), it was observed that LY294002markedly suppressed the tumor-induced angiogenic response in this model(MVD is 22±5 in LY294002 treated tumors versus 50±6 in the controls).Importantly, microvessel determinations were performed on day 7 afterimplantation to compare angiogenic activity of tumors of similar size.At the time of analysis the tumors were approximately 400 mm3 in thecontrol and approximately 35 mm3 in the LY294002 treated mice. Thesedata argue against an effect of tumor hypoxia or size on the inductionof angiogenesis. It is likely that the effects of LY294002 are complexand that the size of tumor mass may contribute at later time points tothe induction of angiogenesis. Despite this caveat, the data demonstratethat LY294002 dramatically suppressed the angiogenic response of U87MGcells in vivo.

[0190] We also examined the effect of LY294002 on tumor growth andincidence of brain tumor in a nude mouse model. See FIG. 17. In aprevious study, in vivo activity of LY294002 against an ovariancarcinoma was observed. However, it is difficult to interpret theseresults as the tumor was grown as an ascitic tumor and LY294002 wasinjected into peritoneal cavity. In other words, the carcinoma was notassessed in its natural milieu. Hu et al., Clin. Can. Res. 6:880). Ourresults show conclusively that subcutaneous tumor growth is markedlysuppressed by LY294002 treatment (FIGS. 15 and 17). In additionalexperiments, we observed that LY294002 markedly suppressed theintracranial growth of U87MG cells in nude mouse model. In control micetreated with DMSO, ⅘ had grossly visible and/or histologically confirmedbrain tumors by H & E analysis by day 25 after implantation, whereas in{fraction (0/5)} mice treated with LY294002 had grossly detectable orhistologic evidence of intracranial tumor when examined on day 42. Otherrecent reports suggest that LY294002 and PTEN reconstitution mayincrease tumor responsiveness to DNA damage from chemotherapy andradiation. Our results suggest that PTEN exerts its regulatory effectsthrough the induction of p53 transcription, a known factor in theinduction of apoptosis.

[0191] Recent in vitro data suggest a link between PI-3 kinase anddownstream targets including AKT, HIF1α and VEGF in the potentialcontrol of angiogenesis. In the present example, we show that the PTENtumor suppressor controls p53 transcription in U87 glioblastoma cells.Holland et al recently reported that the introduction of activated AKTand Ras into glial cells of the mouse brain results in the developmentof glioblastomas (Holland et al., (2000) Nat. Genet. 25:55). Thus, thecombined data indicate that inhibitors of PI-3 kinase and downstreamtargets such as AKT should provide therapeutic efficacy in the treatmentof malignant gliomas. Finally, the results presented herein provide thefirst evidence that LY294002 controls tumor-induced angiogenesis througha mechanism that appears to involve the regulation of p53 transcription.Accordingly, these data support the hypothesis that these two tumorsuppressor genes are localized on the same signaling pathway for thecoordinated control of angiogenesis in tumor cells.

EXAMPLE V PTEN Reconstitution Enhances Sensitivity of Tumors toChemotherapy and Radiation Therapy in vitro and in vivo

[0192] The data presented in Example IV demonstrate that a molecularlink exists between the activities of PTEN and p53 tumor suppressorgenes, p53-mediated transcription and phosphorylation of MDM2 by AKT.These data indicate that activation of the PI-3 kinase pathway can becorrelated with a reduction of programmed cell death leading to a higherrisk of tumor progression. Thus, agents which modulate the PTEN pathwaycan be used in certain cell populations to influence the apoptoticmechanisms triggered by chemotherapy and radiation and other cellularstresses. PI-3 kinase, MDM2 and AKT as well as PTEN provide idealbiological targets for the development of such agents. Thus, the presentinvention provides methods for identifying and biochemicallycharacterizing small molecules which modulate the biological processesregulated by these proteins, including, but not limited to apoptosis,proliferation, differentiation and chemosensitivity.

[0193] The data set forth in this example implicate a role for PTEN inchemo- and radiosensitivity which influences tumor-induced angiogenesisand p53 tumor suppressor activity. Since PTEN is mutated or deleted in50% of all glioblastoma, we assessed whether such cells are renderedsensitive to chemotherapy upon stable introduction of wild-type PTEN.U87MG glioblastoma cells, null for PTEN, or glioblastoma cells stablyexpressing catalytically inactive PTEN (R130M cells), required 4.7 μM ofetoposide to achieve a 50% cell kill. Glioblastoma cells engineered tostably express wild-type PTEN were sensitized requiring 0.047 μM ofetoposide to achieve a 50% cell kill, a 100-fold increase insensitivity. See FIGS. 18 and 19. Compensation for the absence of PTENin the parental line of glioblastoma was achieved by treatment of thecells with LY294002. This agent rendered the parental, PTEN-null cellsas sensitive to etoposide as cells overexpressing PTEN. Theseobservations show that PTEN and inhibitors of PI 3-kinase sensitizecancers to chemotherapy by blockade of PI 3-kinase/Akt survivalsignaling, thereby permitting the p53 tumor suppressor to transmit itsdeath signal.

[0194] The following protocols are provided to facilitate the practiceof Example VI.

[0195] Materials.

[0196] The D0p1 antibody to p53 was from Santa Cruz Biotechnology.

[0197] Cell Culture, Treatments, and Transfections.

[0198] U87MG cells, breast adenocarcinomas, MCF-7 cells, T47D breastductal carcinoma cells, 293T human embryonic kidney cells transformedwith adenovirus, and H1299 non-small lung carcinoma cells were culturedin DMEM supplemented with 10% fetal bovine serum at 37° C. under 5%CO_(2.). Stable clones of U87MG and U373MG glioma cells were generatedusing retroviral vectors pBABEpuro to express wild type and mutants ofPTEN.

[0199] The MDM2 promoter with and without p53 response elements wasoperably linked to a nucleic acid encoding luciferase as described inExample IV. Luciferase assays were conducted twenty-four hours aftertransient transfection using the luciferase assay system and thegalacto-light kit to assay β-gal activity.

[0200] MTT Assays.

[0201] Cells were seeded (6000/well) into a 96 well plate. Twenty-fourhours later the growth medium was changed to 1% serum and other reagentswere added as described. Treatments proceeded for 24-72 h. At each timepoint the MTT assay was performed. MTT (10 ul of a 5 mg/ml stocksolution dissolved in RPMI medium) is added to each well of cultures 4hours before quantitation of viable cell number by measuring absorbancein each well at 650 nm with a microplate reader (Molecular Devises,)

[0202] Agents added to cell LY294002 alone or in combination withetoposide. In addition, comparison was made between U87MG cells null forPTEN and reconstituted with PTEN protein. The absorbance of each wellwas measured at 570 nM with a microplate reader. The amount ofabsorbance is a reflection of mitochondrial activity of cells and henceis a method for the quantitation of viable cell numbers in multiplereplicates.

[0203] Effect of PTEN Expression on Radiosensitivity.

[0204] To determine the role of PI-3 kinase pathways and PTEN in controlof radiation sensitivity, U87MG reconstituted with wild type or mutant(G129R) forms of PTEN under constitutive or muristirone inducedconditions were exposed in vitro to a gamma irradiation source of 12 Gy,20Gy, 30Gy, 40Gy, 50Gy at 1.2Gy per minute as a single dose.

[0205] Cells were then washed gently and plated in tissue culture 96well plate and at different time intervals after radiation exposureviable cell numbers were determined using MTT assay. An IC50 wasestablished for cells expressing PTEN or treated with 5-10 uM LY294002.The results are similar to those observed with etoposide sensitivity inthat PTEN reconstitution or LY294002 dramatically increased theradiation sensitivity of this p53 wild type tumor in vitro. Evaluationin vivo showed similar results as described for our experiments forantiangiogenic and antimetastatic effect of LY294002 or PTENreconstitution.

[0206] The data reveal that the induction of p53 associated with PTENwas operative in the induction of a radiosensitive state.

[0207] The clones utilized in this example express physiologic levels ofPTEN thereby more closely approximating biological reconstitution ofPTEN rather than overexpression of the protein. In the studies describedin the prior art and the previous examples, PTEN is overexpressed andgenerates cells which are growth arrested by virtue of excess levels ofPTEN. PTEN expression was assessed in the present U373MG clones byWestern blot and found to be equal to or less than levels of PTENprotein measured in whole brain lysates or cultivated human primaryastrocytes. Ideally, assays for assessing PTEN function should be basedon experimental conditions wherein PTEN expression levels accuratelyreflect the levels observed in normal cells. It is known that PTENlevels are closely regulated by endogenous biochemical feedbackmechanisms under normal conditions thereby allowing cells to undergoregulated proliferation in response to proper physiological stimuli.Thus, PTEN functions in normal cells as a rheostat to control vital PIP₃linked functions.

[0208] As demonstrated herein, PTEN activity influences the sensitivityof U87MG cells to genotoxic stress induced by the topoisomeraseinhibitor, etoposide (VP16) and radiation. Exposure of U87MG cells toetoposide in the presence of PT-3 kinase inhibitor, LY294002dramatically shifted the dose/response curve towards a more sensitivestate. Similarly PTEN reconstituted tumor cells were markedly moresensitive to etoposide induced cell death and radiation. Thus, PTENactivity modulates p53 function thereby regulating and coordinating p53levels in the cell. Accordingly, activation of PTEN and the PI-3 kinasepathway sensitizes cells to p53 mediated cell death through the controlof p53 induced apoptosis.

[0209] The data reveal that LY294002 dramatically sensitizes theparental U87MG cells to the cytotoxic effect of etoposide (VP-16) asshown by MTT assay at a concentration where LY294002 has no effect oncell viability. The concentration of etoposide utilized demonstratesminimal cytotoxic activity (1 uM) while the combination of etoposide andLY294002 appears to act synergistically giving rise to enhancedcytotoxicity. There is a 5-fold increase in cytotoxicity observed incells pretreated with the PI-3 kinase inhibitor. These results have beenrepeated 20 times and are highly statistically significant (p<0.0001)and show that LY294002 induces a chemosensitive state in this p53 wildtype glioma cell line. Similar data were obtained for effect of PTENreconstitution in this system and in other tumor cell lines. PTENreconstitution or LY294002 induced a marked sensitivity of tumor cellsto etoposide, adriamycin, cytoxan, asparaginase, vincristine, busulfanand other chemotherapeutic agents. In addition LY294002 and PTENreconstitution induce a marked sensitivity to ionizing radiation usingthe same methodology.

[0210] LY294002 and PTEN reconstitution also induce a sensitivity toapoptosis induced by other environmental stress including: osmotic,endotoxic stress, nutritional stress, metabolic stress, hyperoxic,hypoxic, chemical, temperature, immunologic, other forms ofelectromagnetic radiation, heat shock, using the same methods forenablement. The inhibition of PTEN reduced the apoptotic response toeach of these forms of cellular stress in vivo and in vitro.

[0211] While certain preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made to the invention without departing from the scope and spiritthereof as set forth in the following claims.

What is claimed is:
 1. A method for identifying agents which modulatePTEN activity, comprising: a) providing a host cell wherein PTEN isexpressed; b) contacting said host cell with a test agent suspected ofmodulating PTEN activity; c) assessing said modulation as a function ofalterations in activated AKT levels in the presence of said agent.
 2. Amethod as claimed in claim 1, wherein said PTEN is mutated.
 3. A methodas claimed in claim 2, wherein said mutation in PTEN is selected fromthe group consisting of G129E, G129R, R130M and C124S.
 4. A method asclaimed in claim 2, wherein said PTEN is inactive and said test agentsuppresses concomittant AKT activating activity.
 5. A test agentidentified by the method of claim
 4. 6. A method for gene therapy forthe treatment of cancers arising from a mutation in PTEN, comprisingadministration of a nucleic acid encoding wild type PTEN to a patient inneed thereof.
 7. The method as claimed in claim 6, wherein said PTENencoding nucleic acid is inserted into a vector having tropism for saidcancer cell.
 8. The method as claimed in claim 7, wherein said vector isselected from the group consisting of retroviral vectors, adenoviralvectors, shuttle vectors, disabled vaccinia viral vectors, and plasmidvectors.
 9. The method as claimed in claim 8, wherein said plasmidvector is encased in an antibody studded liposome, said antibody beingimmunologically specific for an antigen present on a tumor cell.
 10. Themethod as claimed in claim 9, wherein said tumor cell is a glioma celland said antigen is the epidermal growth factor receptor.
 11. A methodas claimed in claim 10, wherein said liposome is a cationic liposome.12. A method as claimed in claim 7, wherein said vector disposed in abiologically compatible medium is microinjected directly into saidcancer cell.
 13. A method for identifying agents which modulate PTENangiogenic activity, comprising: a) providing a host cell wherein PTENis expressed; b) contacting said host cell with a test agent suspectedof modulating PTEN angiogenic activity; c) assessing said modulation asa function of alterations in microvessel density formation in thepresence of said agent.
 14. A method as claimed in claim 13, whereinsaid PTEN is mutated.
 15. A method as claimed in claim 14, wherein saidmutation in PTEN is selected from the group consisting of G129E, G129R,R130M and C124S.
 16. A method as claimed in claim 13, wherein said PTENis inactive and said test agent inhibits PTEN mediated angiogenicactivity.
 17. A method as claimed in claim 13, wherein said microvesseldensity formation determined via immunospecific binding of anti-CD31antibodies.
 18. A test agent identified by the method of claim
 16. 19. Amethod for identifying agents which modulate PTEN activity, comprising:a) providing a host cell wherein PTEN is expressed; b) contacting saidhost cell with a test agent suspected of modulating PTEN activity; c)assessing said modulation as a function of alterations in upregulationof TSP-1 in the presence of said agent.
 20. A method as claimed in claim19, wherein said PTEN is mutated.
 21. A method as claimed in claim 20,wherein said mutation in PTEN is selected from the group consisting ofG129E, G129R, R130M and C124S.
 22. A method as claimed in claim 20,wherein said PTEN is inactive and said test agent restores PTEN mediatedupregulation of TSP-1.
 23. A test agent identified by the method ofclaim
 22. 24. A method for identifying agents which modulate PTENactivity, comprising: a) providing a host cell wherein PTEN isexpressed; b) contacting said host cell with a test agent suspected ofmodulating PTEN activity; c) assessing said modulation as a function ofalterations in VEGF levels in the presence of said agent.
 25. A methodas claimed in claim 24, wherein said PTEN is mutated.
 26. A method asclaimed in claim 25, wherein said mutation in PTEN is selected from thegroup consisting of G129E, G129R, R130M and C124S.
 27. A method asclaimed in claim 26, wherein said PTEN is inactive and said test agentrestores regulated PTEN inhibition of VEGF production.
 28. A test agentidentified by the method of claim
 27. 29. A method for identifyingagents which modulate PTEN activity, comprising: a) providing a hostcell wherein PTEN is expressed; b) contacting said host cell with a testagent suspected of modulating PTEN activity; c) assessing saidmodulation as a function of alterations in TIMP3 levels in the presenceof said agent.
 30. A method as claimed in claim 29, wherein said PTEN ismutated.
 31. A method as claimed in claim 30, wherein said mutation inPTEN is selected from the group consisting of G129E, G129R, R130M andC124S.
 32. A method as claimed in claim 30, wherein said PTEN isinactive and said test agent restores PTEN mediated induction of TIMP3.33. A test agent identified by the method of claim
 32. 34. A method foridentifying agents which modulate PTEN activity, comprising: a)providing a host cell wherein PTEN is expressed; b) contacting said hostcell with a test agent suspected of modulating PTEN activity; c)assessing said modulation as a function of alterations in MMP-9 levelsin the presence of said agent.
 35. A method as claimed in claim 36,wherein said PTEN is mutated.
 36. A method as claimed in claim 35,wherein said mutation in PTEN is selected from the group consisting ofG129E, G129R, R130M and C124S.
 37. A method as claimed in claim 36,wherein said PTEN is inactive and said test agent restores regulatedPTEN suppression of MMP9 activity.
 38. A test agent identified by themethod of claim
 37. 39. A method for identifying agents which modulatePTEN activity, comprising: a) providing a host cell wherein PTEN isexpressed; b) contacting said host cell with a test agent suspected ofmodulating PTEN activity; c) assessing said modulation as a function ofalterations of invasiveness of said cells in the presence of said agent.40. A method as claimed in claim 39, wherein said PTEN is mutated.
 41. Amethod as claimed in claim 39, wherein said mutation in PTEN is selectedfrom the group consisting of G129E, G129R, R130M and C124S.
 42. A methodas claimed in claim 40, wherein said PTEN is inactive and said testagent restores PTEN mediated inhibition of invasiveness.
 43. A testagent identified by the method of claim
 42. 44. A method for identifyingan agent which modulates PTEN phosphatase activity comprising: a)providing an enzymatically active PTEN molecule or peptide fragment in abiological buffer; b) adding to said buffer a substrate of said PTENenzyme, said enzymatic action of PTEN on said substrate giving rise to adetectable reaction product; c) contacting said active PTEN molecule orfragment in said biological buffer with an agent suspected of modulatingsaid PTEN phosphatase activity; d) measuring said PTEN phosphataseactivity in the presence and absence of said agent.
 45. A method asclaimed in claim 44, wherein said PTEN is immobilized on a solidsupport.
 46. A method as claimed in claim 45, wherein said PTEN activityis assessed in a high throughput format.
 47. A method as claimed inclaim 44, wherein said PTEN activity is contacted with a plurality oftest agents.
 48. A method as claimed in claim 44, wherein a Malachitegreen assay is performed to determine said enzymatic activity of PTEN inthe presence and absence of said test agent.
 49. A method foridentifying agents having binding affinity for PTEN or peptide fragmentthereof, said method comprising: a) providing a PTEN molecule or peptidefragment in a biological buffer; b) contacting said PTEN molecule orfragment in said biological buffer with a detectably labeled agentsuspected of having binding affinity for said PTEN or peptide fragmentthereof, such that a detectably labeled complex forms between thoseagents having affinity for said PTEN or fragment thereof; and d)identifying and isolating said detectably labeled complex if present,thereby identifying said agent.
 50. A method as claimed in claim 49,wherein said PTEN or PTEN peptide fragment is adsorbed to a solidsupport.
 51. A method as claimed in claim 50, wherein said method isperformed in a high throughput screening format.
 52. A method as claimedin claim 49, wherein said PTEN or fragment thereof is contacted with aplurality of detectably labeled agents present in a chemicalcombinatorial library.
 53. A method as claimed in claim 49, wherein saidPTEN or PTEN fragment is expressed on the surface of a phage, and saidexpressed PTEN or PTEN fragment is contacted with a plurality ofdetectably labeled agents present in a chemical combinatorial library.54. A method as claimed in claim 49, wherein said PTEN fragment consistsessentially of a fragment selected from the group consisting ofDLDLTYIYP (SEQ ID NO: 3), YLVLTL (SEQ ID NO: 6), YRNNIDD (SEQ ID NO: 8),KGVTIPSQRRYVYYYSYLL (SEQ ID NO: 15), YSYL (SEQ ID NO: 7), YFSPN (SEQ IDNO: 5), RYSDTTDS (SEQ ID NO: 16), HCKAGKR (SEQ ID NO: 9), DHNPPQ (SEQ IDNO: 10), KGVTIPSQRRY SEQ ID NO: 17), HFWVNTFFI (SEQ ID NO: 11), TLTKNDLD. . . FTKTV (SEQ ID NO: 12), GDIKVEF . . . FTKTV (SEQ ID NO: 13),DKANKDKAN . . . FTKTV (SEQ ID NO: 14), and HTQITKV (SEQ ID NO: 18). 55.A method as claimed in claim 53, wherein said phage express a PTENfragment consisting essentially of a fragment selected from the groupconsisting of DLDLTYIYP (SEQ ID NO: 3), YLVLTL (SEQ ID NO: 6), YRNNIDD(SEQ ID NO: 8), KGVTIPSQRRYVYYYSYLL (SEQ ID NO: 15), YSYL (SEQ ID NO:7), YFSPN (SEQ ID NO: 5), RYSDTTDS (SEQ ID NO: 16), HCKAGKR (SEQ ID NO:9), DHNPPQ (SEQ ID NO: 10), KGVTIPSQRRY (SEQ ID NO: 15), HFWVNTFFI (SEQID NO: 11), TLTKNDLD . . . FTKTV (SEQ ID NO: 12), GDIKVEF . . . FTKTV(SEQ ID NO: 13), DKANKDKAN . . . FTKTV SEQ ID NO: 14), and HTQITKV (SEQID NO: 18).
 56. A method for preventing or inhibiting inflammatorydisease in a patient in need thereof, comprising the administration ofan effective amount of a PTEN agonist.
 57. A method as claimed in claim56, wherein said inflammatory disease is selected from the groupconsisting of macular degeneration, arthritis, asthma, hay fever,systemic lupus erythrematosis, Crohn's disease, and inflammatory boweldisease.
 58. A method as claimed in claim 56, further comprising theadministration of an inhibitor of PI-kinase.
 59. A method as claimed inclaim 58, wherein said PI-kinase inhibitor is LY294002.
 60. A method asclaimed in claim 56, further comprising the administration of an AKTinhibitor.
 61. A method for the treatment of cancer in a patient in needthereof, comprising the administration of an effective amount of a PTENagonist.
 62. A method as claimed in claim 61, wherein said PTEN agonisteffectively blocks cancer cell metastasis.
 63. A method as claimed inclaim 61, wherein said PTEN agonist effectively blocks angiogenesis. 64.A method as claimed in claim 61, further comprising the administrationof at least one additional chemotherapeutic agent.
 65. A method asclaimed in claim 64, wherein said at least one additionalchemotherapeutic agent is selected from the group consisting ofalkylating agents, antimetabolites, asparaginase, vincristine,vinblastine, anthracyclines, microtubule disrupting agents, taxol,herceptin, and etoposides.
 66. A method as claimed in claim 61, furthercomprising the administration of an inhibitor of PI3 kinase.
 67. Amethod as claimed in claim 66, wherein said PI-kinase inhibitor isLY294002.
 68. A method as claimed in claim 61, further comprising theadministration of an inhibitor of AKT.
 69. A method for inhibiting p53mediated programmed cell death in a patient in need thereof, said methodcomprising the administration of a PTEN inhibitor.
 70. A method forenhancing the chemosensitivity of tumor cells in a patient in needthereof, said method comprising the administration of an PTEN agonist toa patient having a chemoresistant tumor.
 71. A method for enhancing theradiosensitivity of a tumor cells in a patient in need thereof, saidmethod comprising the administration of a PTEN agonist to a patienthaving a radioresistant tumor.
 72. A method of gene therapy for thetreatment of an inflammatory condition in a patient having a mutation inPTEN, said method comprising delivery of a native PTEN encoding nucleicacid to immune cells of said patient.
 73. A method as claimed in claim72, further comprising the administration of a PI3 kinase inhibitor. 74.A method as claimed in claim 72, wherein said immune cell is selectedfrom the group consisting of mast cells, B cells, T cells, dendriticcells, neutrophils, eosinophils and macrophages.
 75. A method forinhibiting immunoreceptor signaling in a patient in need thereof,comprising administration of an effective amount of a PTEN agonist. 76.A method as claimed in claim 75, wherein said immunoreceptor is selectedfrom the group consisting of a T cell receptor, B cell receptor,ITAM-bearing receptor, FcγR, FcεR, and FcαR.
 77. A method as claimed inclaim 75, wherein said agonist is administered to prevent a conditionselected from the group consisting of graft rejection and graft versushost disease.
 78. A method for augmenting an immune reaction in apatient in need thereof, comprising administration of an effectiveamount of an inhibitor of PTEN.
 79. A method as claimed in claim 78,wherein said inhibitor is targeted to a cell selected from the groupconsisting of T cells, B cells, and macrophages.
 80. A method forinhibiting aberrant angiogenesis in a patient in need thereof, saidmethod comprising the administration of a PI3 kinase inhibitor.
 81. Amethod as claimed in claim 80, wherein said aberrant angiogenesis iscaused by cancer, autoimmune disease, arthritis, systemic lupuserthymatosis, inflammatory bowel disease, coronary artery disease,cerebrovascular disease, and atherosclerosis.
 82. A method as claimed inclaim 80, further comprising the administration of an AKT inhibitor. 83.A method for inhibiting aberrant angiogenesis in a patient in needthereof, said method comprising the administration of an AKT inhibitor.84. A method as claimed in claim 83, wherein said aberrant angiogenesisis caused by cancer, autoimmune disease, arthritis, systemic lupuserthymatosis, inflammatory bowel disease, coronary artery disease,cerebrovascular disease, and atherosclerosis.
 85. A method as claimed inclaim 83, further comprising the administration of an PI3 kinaseinhibitor.
 86. A method for inhibiting p53 mediated programmed celldeath in a patient in need thereof, comprising the targetedadministration of a PTEN inhibitor to normal tissues to inhibit stressinduced apoptosis thereof, wherein said patient is in need for suchtreatment due to a condition selected from the group consisting ofmyocardial infarction, cerebrovascular insult and gram negative sepsis.87. A method for inhibiting p53 mediated programmed cell death in apatient in need thereof, comprising the targeted administration of aPTEN inhibitor, said PTEN inhibitor inhibiting cellular senescencethereby promoting survival of normal cells.
 88. A method as claimed inclaim 87, wherein said normal cells are selected from the groupconsisting of brain cells, heart cells, and skin cells.